Name: a detailed protocol for characterizing the murine c1498 cell line and its associated leukemia mouse model
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Description: Watch this Scientific Journal Video about A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model at JoVE.com

The authors would like to acknowledge the "Ligue Nationale contre le Cancer" (Comit&#233; du Septentrion), the SIRIC ONCOLille (Grant INCa-DGOS-INSERM 6041) and the Institut pour la Recherche sur le Cancer de Lille (IRCL) for supporting this work. They would like to thank Delphine Taillieu and the animal facility staff for housing the mice and maintaining their welfare. We also thank Rapha&#235;lle Caillerez and Nathalie Jouy for their respective help in microscopy and flow cytometry.

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The authors declare that they have no competing financial interests.

In studi precedenti, la linea cellulare C1498 è stato descritto come un induttore di granulocitica acuta 5, mielomonocitica 6 o 7 NKT leucemia a cellule. Tuttavia, i dati dimostrativi in ​​letteratura erano assenti o incompleti. Il protocollo qui presentata utilizza tecniche diverse, come la citometria a flusso, immunofluorescenza, MGG colorazione e saggi citochimiche, per caratterizzare le cellule in coltura e C1498 per determinare la natura della leucemia che è indotta nei topi dopo che sono stati iniettati. Quando abbiamo phenotyped in cellule C1498 vitro in coltura dopo immunostaining e citometria a flusso sono state eseguite le analisi, abbiamo osservato alcune limitazioni, perché queste cellule hanno espresso alcuni marcatori di superficie delle cellule ematopoietiche che sono stati precedentemente descritti in letteratura 6,7. In accordo con i nostri risultati, Labelle et al., Non ha rispettato l&#39;espressione di superficie delle cellule del maturo TCR sulle cellule C1498 con cytomet flussory colorazione. Tuttavia, li hanno considerati come una linea di cellule NKT dopo aver rilevato CD3ε e TCRVβ8.2 mRNA 7. Abbiamo anche osservato l&#39;espressione intracellulare di catene TCRVβ e molecole CD3ε nella maggior parte delle cellule (&gt; 70%), ma le loro linee ematopoietiche non è stato possibile determinare perché c&#39;era anche concomitante espressione intracellulare del Mac-3 molecola. Mieloperossidasi, MGG colorazione e valutazioni per analizzare esterasi funzionali utilizzando cytochemistry dimostrato che la linea cellulare C1498 ha avuto un&#39;origine mieloide e era composto da monoblasti e mieloblasti. Questi risultati erano in accordo con la percentuale di cellule Mac-3 + che sono stati ottenuti in citometria a flusso colorazione. Sebbene non quantitativa, questi passaggi rappresentano esperimenti chiave da eseguire. In effetti, essi rimangono, fino ad ora, i migliori metodi esistenti per la caratterizzazione del lignaggio e la differenziazione fase di cellule ematopoietiche che esprimono no o Fmarcatori fenotipici specifici Ew. Citometria a flusso colorazione è stato utile per dimostrare lo sviluppo di leucemia acuta nei topi congenic dopo C1498 cellule sono state iniettate per via endovenosa. I CD45.2 + C1498 cellule che si infiltrarono nel sangue periferico e vari organi sono stati isolati, e le loro frequenze sono state determinate. La quantificazione è stata effettuata anche per analizzare midollare intrinseca e le cellule della milza dopo immunofenotipizzazione. Sono stati rilevati Limitazioni quando il fenotipo cellulare C1498 è stata esaminata in organi come espressi alcuni marcatori ematopoietici (solo alcuni di loro erano B220 +). Per definire la natura della leucemia acuta osservato, May-Grünwald Giemsa e un&#39;analisi delle attività di monocitica e esterasi granulocitari state eseguite utilizzando midollo osseo. I risultati hanno mostrato che le cellule C1498 conservato la loro morfologia e la funzione mieloblastica e monoblastica, rivelando l&#39;insorgenza della leucemia mielomonocitica. <p class = &quot;jove_content&quot;&gt; In considerazione delle fasi critiche descritte in questo protocollo, particolare attenzione dovrebbe essere data a pH durante l&#39;esecuzione di reazioni citochimiche e MGG colorazione perché gli errori di pH possono portare a interpretazioni non corrette dei risultati. Per esempio, α-naftil butirrato esterasi attività è specifica per le cellule monocitiche solo a pH di 6,0 perché granulociti e linfociti possono macchiare positivo per questo test a valori di pH superiori. Fissarsi le cellule non è raccomandato prima di eseguire MGG colorazione, e ci ha mostrato che solo la fissazione CAF ha fornito risultati soddisfacenti durante l&#39;esecuzione di esterasi reazioni citochimiche utilizzando C1498 cellule. Per preservare l&#39;espressione della molecola CD115 e la sua individuazione mediante citometria a flusso, tutti i campioni (cellule esempio, sangue, midollo osseo e milza) deve essere mantenuto in ghiaccio durante la procedura. Se nessuna colorazione si osserva in citometria a flusso o / e immunofluorescenza, il riferimento degli anticorpi, loro immagazzinamento reencomi e le loro diluizioni devono essere controllati. I riferimenti indicati nella tabella materiali / attrezzature sono state selezionate per le applicazioni citometria di flusso o di immunofluorescenza. Le primarie / anticorpi secondari o loro fluorofori coniugati potrebbero aver perso la loro attività a causa di stoccaggio inadeguato (ad esempio, l&#39;esposizione alla luce o al calore), la diluizione improprio, vasta di congelamento / scongelamento o l&#39;uso di tamponi contaminati. Eseguire controlli positivi per garantire che funzionino correttamente. Usa il mouse midollo osseo o cellule della milza-derivati ​​che sono noti per esprimere le proteine ​​di interesse. Per evitare di fondo e marcatura non specifica, fare in modo che le cellule vengono lavate correttamente e mantenuti a elevata umidità (per immunofluorescenza) e che gli anticorpi sono diluiti secondo le istruzioni. Utilizzare la stessa concentrazione e diluizione per l&#39;anticorpo di controllo isotipo e l&#39;anticorpo primario per determinare con precisione il livello di fondo nel campione. Per gli esperimenti esterasi citochimica, lareagenti possono essere testati utilizzando vetrini di controllo positivi e negativi, che contengono il mouse purificato granulocitica della milza (Ly6G +) e monocitica (+ CD115), le cellule. La procedura descritta in questo studio ha dimostrato che molte delle caratteristiche leucemici osservati nei topi dopo l&#39;iniezione di cellule C1498 condiviso caratteristiche comuni con umana leucemia mielomonocitica acuta 11,12. Le cellule leucemiche invase comportato una riduzione di mature e immature (progenitori e precursori) cellule ematopoietiche midollari. cellule C1498 sono presenti a frequenza elevata (&gt; 20%) nel sangue periferico, come lo sono le cellule monociti. Epatomegalia e splenomegalia sono stati osservati per derivare dalla infiltrazione di cellule leucemiche, e aumenti significativi linfociti B e delle cellule mieloidi sono state osservate anche per accompagnare splenomegalia. Trombocitopenia è stata osservata anche quando i numeri delle piastrine del sangue sono stati stimati utilizzando un analizzatore ematologico. E &#39;stato spettacolon, con esperimenti in vitro, che C1498 cellule inibiscono la normale emopoiesi murina secernendo fattori solubili 13. In diversi modelli murini di tumore, le cellule mieloidi immature (tra cui monociti e cellule granulocitari) hanno anche dimostrato di migrare dal midollo osseo alla milza, dove inibiscono l&#39;attivazione delle cellule T specifiche anti-tumorale e la proliferazione 14. Pertanto, la riduzione in cellule ematopoietiche che è stato osservato nel midollo osseo potrebbe essere il risultato di una carenza sia in ematopoiesi e / o dal loro emigrazione. Quest&#39;ultimo meccanismo potrebbe spiegare la presenza di monocitosi nel sangue periferico o l&#39;osservazione di frazioni mieloidi ingrandita nella milza. E &#39;anche ipotizzabile che queste cellule avrebbero potuto essere derivato dal miglioramento della emopoiesi della milza. Infatti, in condizioni stazionarie, alcuni sottoinsiemi di cellule B della milza sono stati identificati come precursori di linfociti maturi B 15. Inoltre, in condizioni infiammatorie, medLe cellule staminali midollare e progenitori hanno dimostrato di trasferirsi alla milza per indurre la produzione di monociti maturi 16. Questo protocollo non ci permette di trarre conclusioni per quanto riguarda i meccanismi che sono coinvolti nello sviluppo della leucemia, e saggi funzionali nonché molecolari supplementari dovrebbero essere impiegate per farlo. Tuttavia, questi dati includono informazioni dettagliate sulle caratteristiche cliniche della leucemia mielomonocitica acuta e aiuteranno i ricercatori a valutare e comprendere gli effetti dei nuovi agenti terapeutici.

leucemia mieloide acuta (LMA) è caratterizzata dalla proliferazione incontrollata di cellule mieloidi ematopoietiche che sono bloccati in diversi stadi di maturazione. Questo disregolazione può influenzare il granulociti, monociti, eritrocitaria o percorsi di differenziazione megaryocytic 1. cellule AML si accumulano nel midollo osseo, con conseguente compromissione emopoiesi, che si traduce in trombocitopenia, linfopenia e anemia. Le cellule leucemiche anche invadere il sangue e gli organi non linfoidi. Il modello C1498 del mouse è stato utilizzato per decenni come modello per la leucemia acuta poiché le cellule tumorali sono stati isolati da un leucemico 10 mesi C57BL / 6 (H-2 b) topo femmina nel 1941. La letteratura descrive l&#39;invasione nel sangue , gli organi ematopoietici (ad esempio, i milza e nei linfonodi) e gli organi non ematopoietici (ad esempio, il fegato, i polmoni, ovaie e reni) da parte delle cellule C1498 altamente proliferative dopo che sono stati iniettati attraverso un aendovenosa, sottocutanea o via intraperitoneale in topi suscettibili 2-4. Tuttavia, questo modello di topo è stato segnalato per indurre sia granulocitica 2,5 o 6 mielomonocitica leucemia. Più recentemente, uno studio pubblicato nel 2002 ha descritto questo tipo di cancro, come la leucemia cellule murine NKT 7. Così, letteratura differisce circa la natura di questa linea cellulare C1498 e leucemia associato induce nei topi. Queste discrepanze sono dovute principalmente alla mancanza di dettagliate e aggiornate informazioni pubblicate sulle cellule e la malattia leucemica in generale, perché molti studi sono stati effettuati nel 1950 - &#39;70. Qui forniamo un protocollo dettagliato per descrivere come caratterizzare cellule C1498 e analizzare la natura della malattia leucemica che è indotta dalla loro iniezione endovenosa in topi. La prima sezione di questo protocollo è dedicato ad una descrizione di cellule C1498 che sono stati coltivati in vitro. contro fluorescentecorpi diretti contro la superficie e marcatori ematopoietici intracellulari sono stati usati per determinare il loro fenotipo mediante citometria a flusso. La presenza di mieloperossidasi è stata valutata utilizzando la microscopia di immunofluorescenza, la loro stirpe ematopoietico e differenziazione fase sono stati valutati utilizzando citochimica per valutare l&#39;attività di esterasi, ed è stata effettuata maggio-Grünwald Giemsa. Le cellule C1498 sono state iniettate in topi, e la malattia leucemia acuta che è stata indotta è descritto nella seconda sezione di questo manoscritto. Le stesse tecniche sono stati usati per determinare le frequenze ed i fenotipi di cellule leucemiche e inerenti nel midollo osseo, sangue periferico, la milza e gli organi non ematopoietici (il fegato e polmoni). Questo protocollo è altamente riproducibile, ed i dati presentati qui aiuterà i ricercatori a valutare gli effetti di nuove strategie terapeutiche. Questo modello leucemia del mouse è già stato utilizzato per testare immunoterapia avvicina unnd diversi farmaci chemioterapici cancro 8,9. La loro efficacia è stata valutata determinando l&#39;evoluzione del carico tumorale e il tasso di sopravvivenza. Questo protocollo può essere utilizzato per fornire ulteriori informazioni sulla distribuzione e la sussistenza delle popolazioni di cellule ematopoietiche leucemiche e altri durante il trattamento.

<table><tbody><tr><td>C1498 cell line</td><td>ATCC</td><td>TIB 49</td><td></td></tr><tr><td>C57BL/6J-Ly5.1</td><td>Charles River</td><td>B6.SJL-Ptprc a Pep3 b/BoyCrl</td><td></td></tr><tr><td>&lt;strong&gt;Cells culture reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Mercaptoethanol, Gibco</td><td>ThermoFisher Scientific</td><td>21985</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher Scientific</td><td>10270</td><td></td></tr><tr><td>HEPES, Gibco (1 M)</td><td>ThermoFisher Scientific</td><td>15630</td><td></td></tr><tr><td>Non-Essential Amino Acids Solution, Gibco&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>11140</td><td></td></tr><tr><td>Penicillin-Streptomycin, Gibco&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>15140</td><td></td></tr><tr><td>RPMI 1640 Medium (Gibco, GlutaMAX Supplement)</td><td>ThermoFisher Scientific</td><td>61870</td><td></td></tr><tr><td>Sodium Pyruvate, Gibco (100 mM)</td><td>ThermoFisher Scientific</td><td>11360</td><td></td></tr><tr><td>&lt;strong&gt;Flow cytometry staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-mouse B220 APC (1)</td><td>ebiosciences</td><td>17-0452</td><td>clone RA3-6B2, final dilution 1/100</td></tr><tr><td>anti-mouse B220 biotin (2)</td><td>ebiosciences</td><td>13-0452</td><td>clone RA3-6B2, 1/400</td></tr><tr><td>anti-mouse CD3 eFluor450 (1)</td><td>ebiosciences</td><td>48-0032</td><td>clone 17A2, 1/100</td></tr><tr><td>anti-mouse CD3 PE (2)</td><td>BD biosciences</td><td>555275</td><td>clone 17A2, 1/100</td></tr><tr><td>anti-mouse CD3 PE-Cy5 (3)</td><td>ebiosciences</td><td>15-0031</td><td>clone 145-2C11, 1/100</td></tr><tr><td>anti-mouse CD4 APC (1)</td><td>ebiosciences</td><td>17-0041</td><td>clone GK1.5, 1/500</td></tr><tr><td>anti-mouse CD4 PE (2)</td><td>ebiosciences</td><td>12-0041</td><td>clone GK1.5, 1/200</td></tr><tr><td>anti-mouse CD8 biotin (1)</td><td>ebiosciences</td><td>13-0081</td><td>clone 53-6.7, 1/100</td></tr><tr><td>anti-mouse CD8 eFluor450 (2)</td><td>ebiosciences</td><td>48-0081</td><td>clone 53-6.7, 1/500</td></tr><tr><td>anti-mouse CD11a biotin</td><td>ebiosciences</td><td>13-0111</td><td>clone M17/4, 1/100</td></tr><tr><td>anti-mouse CD11b PE&amp;nbsp;</td><td>ebiosciences</td><td>12-0112</td><td>clone M1/70, 1/200</td></tr><tr><td>anti-mouse CD16/32 biotin</td><td>ebiosciences</td><td>13-0161</td><td>clone 93, 1/400</td></tr><tr><td>purified anti-mouse CD16/32 (FcR blocking)&amp;nbsp;</td><td>BD biosciences</td><td>553141</td><td>clone 2.4G2</td></tr><tr><td>anti-mouse CD18 biotin (1)</td><td>ebiosciences</td><td>13-0181</td><td>clone M18/2, 1/100</td></tr><tr><td>anti-mouse CD18 FITC (2)</td><td>ebiosciences</td><td>11-0181</td><td>clone M18/2, 1/50</td></tr><tr><td>anti-mouse CD19 PE</td><td>ebiosciences</td><td>12-0193</td><td>clone 1D3, 1/200</td></tr><tr><td>anti-mouse CD21/35 PE</td><td>ebiosciences</td><td>12-0211</td><td>clone 8D9, 1/50</td></tr><tr><td>anti-mouse CD31 PE</td><td>ebiosciences</td><td>12-0311</td><td>clone 390, 1/100</td></tr><tr><td>anti-mouse CD34 eFluor660</td><td>ebiosciences</td><td>50-0341</td><td>clone RAM34, 1/20</td></tr><tr><td>anti-mouse CD44 PE</td><td>BD biosciences</td><td>553134</td><td>clone IM7, 1/50</td></tr><tr><td>anti-mouse CD45.2 FITC&amp;nbsp;</td><td>ebiosciences</td><td>11-0454</td><td>clone 104, 1/100</td></tr><tr><td>anti-mouse CD49b/Pan NK PE</td><td>&amp;nbsp;BD biosciences</td><td>553858</td><td>clone DX5, 1/50</td></tr><tr><td>anti-mouse CD80 biotin</td><td>ebiosciences</td><td>13-0801</td><td>clone 16-10A1, 1/200</td></tr><tr><td>anti-mouse CD86 biotin</td><td>ebiosciences</td><td>13-0862</td><td>clone GL1, 1/200</td></tr><tr><td>anti-mouse CD107b (Mac-3) PE</td><td>ebiosciences</td><td>12-5989</td><td>clone M3/84, 1/40</td></tr><tr><td>anti-mouse CD115 PE</td><td>ebiosciences</td><td>12-1152</td><td>clone AFS98, 1/100</td></tr><tr><td>anti-mouse CD117 eFluor450</td><td>ebiosciences</td><td>48-1171</td><td>clone 2B8, 1/100</td></tr><tr><td>anti-mouse CD127 PE</td><td>ebiosciences</td><td>12-1271</td><td>clone A7R34, 1/100</td></tr><tr><td>anti-mouse CD150 APC</td><td>ebiosciences</td><td>17-1501</td><td>clone 9D1, 1/20</td></tr><tr><td>anti-mouse CD274 (PD-L1) biotin</td><td>ebiosciences</td><td>13-5982</td><td>clone MIH5, 1/200</td></tr><tr><td>anti-mouse Ly-6A/E (Sca-1) PE</td><td>ebiosciences</td><td>12-5981</td><td>clone D7, 1/100</td></tr><tr><td>anti-mouse Ly-6C APC</td><td>ebiosciences</td><td>17-5932</td><td>clone HK1.4, 1/200</td></tr><tr><td>anti-mouse Ly-6G&amp;nbsp; (Gr-1) biotin (1)</td><td>ebiosciences</td><td>13-5931</td><td>clone RB6-8C5, 1/400</td></tr><tr><td>anti-mouse Ly-6G FITC (2)</td><td>ebiosciences</td><td>11-9668</td><td>clone 1A8, 1/50</td></tr><tr><td>anti-mouse MHC class I (H-2Db) biotin</td><td>ebiosciences</td><td>13-5999</td><td>clone 28-14-8, 1/50</td></tr><tr><td>anti-mouse MHC class II (I-A/I-E) PE-Cy5</td><td>ebiosciences</td><td>15-5321</td><td>clone M5/114.15.2, 1/1000</td></tr><tr><td>anti-mouse NK1.1 PE</td><td>BD biosciences</td><td>557391</td><td>clone PK136, 1/50</td></tr><tr><td>anti-mouse TCRVb FITC</td><td>BD biosciences</td><td>553170</td><td>clone H57-597, 1/50</td></tr><tr><td>streptavidin PE-Cy5</td><td>ebiosciences</td><td>15-4317</td><td>&amp;nbsp;&amp;nbsp; 1/200</td></tr><tr><td>Immunofluorescence staining reagents</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse myeloperoxidase (heavy chain antibody)&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>sc-16129</td><td>1/10 (20 &amp;micro;g/ml)</td></tr><tr><td>&amp;nbsp;anti-goat IgG (Texas Red coupled antibody)</td><td>Jackson Immunoresearch</td><td>705-076-147</td><td>1/250</td></tr><tr><td>Normal donkey serum</td><td>Jackson Immunoresearch</td><td>017-000-001</td><td></td></tr><tr><td>Hoechst solution</td><td>BD Biosciences</td><td>561908</td><td>1/1000</td></tr><tr><td>Mounting medium 1 (Fluoromount)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F4680</td><td></td></tr><tr><td>Acetone</td><td>VWR&amp;nbsp;</td><td>20066</td><td></td></tr><tr><td>Methanol</td><td>Merck Millipore</td><td>106009</td><td></td></tr><tr><td>&lt;strong&gt;Esterase cytochemical staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Naphtol AS-D chloroacetate solution</td><td>Sigma-Aldrich</td><td>911</td><td></td></tr><tr><td>Dye solution (Fast Red Violet LB Base solution)</td><td>Sigma-Aldrich</td><td>912</td><td></td></tr><tr><td>pH6.3 buffer concentrate (TRIZMAL)</td><td>Sigma-Aldrich</td><td>913</td><td></td></tr><tr><td>Sodium nitrite solution</td><td>Sigma-Aldrich</td><td>914</td><td></td></tr><tr><td>Citrate solution</td><td>Sigma-Aldrich</td><td>915</td><td></td></tr><tr><td>Hematoxylin solution</td><td>Sigma-Aldrich</td><td>GHS3</td><td></td></tr><tr><td>Acetone</td><td>VWR&amp;nbsp;</td><td>20066</td><td></td></tr><tr><td>Formaldehyde solution, 37%</td><td>Sigma-Aldrich</td><td>F1635</td><td></td></tr><tr><td>&amp;alpha;-naphthyl butyrate solution</td><td>Sigma-Aldrich</td><td>1801</td><td></td></tr><tr><td>Phosphate buffer solution</td><td>Sigma-Aldrich</td><td>1805</td><td></td></tr><tr><td>Sodium nitrite Tablet</td><td>Sigma-Aldrich</td><td>1809</td><td></td></tr><tr><td>Pararosaniline solution</td><td>Sigma-Aldrich</td><td>1804</td><td></td></tr><tr><td>Methylene blue solution</td><td>Sigma-Aldrich</td><td>1808</td><td>1/10</td></tr><tr><td>mounting medium 2 (Clearmount)</td><td>Invitrogen</td><td>00-8010</td><td></td></tr><tr><td>&lt;strong&gt;May-Gr&amp;uuml;nwald Giemsa staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>May-Gr&amp;uuml;nwald solution</td><td>RAL Diagnostics</td><td>320070</td><td></td></tr><tr><td>Giemsa R solution&amp;nbsp;</td><td>RAL Diagnostics</td><td>320310</td><td>&amp;nbsp;&amp;nbsp; 1/20</td></tr><tr><td>pH 6.8 buffer solution</td><td>RAL Diagnostics</td><td>330368</td><td></td></tr><tr><td>&lt;strong&gt;Others materials, reagents and equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ketamine 1000 (100 mg/ml)</td><td>VIRBAC</td><td>3597132111010</td><td></td></tr><tr><td>Xylazine SEDAXYLAN (20 mg/ml)</td><td>CEVA</td><td></td><td></td></tr><tr><td>Bovin albumin serum (BSA) powder</td><td>ThermoFisher Scientific</td><td>BP671</td><td></td></tr><tr><td>PBS solution (1x concentrate)</td><td>ThermoFisher Scientific</td><td>14190</td><td></td></tr><tr><td>Paraformaldehyde (PFA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Saponin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>Ultra Pure 0.5 M EDTA solution, pH 8.0</td><td>ThermoFisher Scientific</td><td>15575</td><td></td></tr><tr><td>Separating solution (Pancoll Mouse)</td><td>PANBIOTECH</td><td>P04-64100</td><td></td></tr><tr><td>Lysis buffer (Red Blood Cells Lysing Buffer) (10x)</td><td>BD Biosciences</td><td>555899</td><td>1/10</td></tr><tr><td>NaOH 10 N</td><td>ThermoFisher Scientific</td><td>SS267</td><td></td></tr><tr><td>Trypan blue solution (0.4 %)</td><td>ThermoFisher Scientific</td><td>15250-061</td><td>1/2</td></tr><tr><td>50 ml tube (Falcon)</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>70 &amp;micro;m Cell Strainer (Falcon)&amp;nbsp;</td><td>Corning Life Sciences</td><td>352350</td><td></td></tr><tr><td>&amp;nbsp;Chamber &amp;amp; filter card (EZ Cytofunnel Shandon)</td><td>Thermo Scientific</td><td>A78710003</td><td></td></tr><tr><td>Microscope Cover Glasses, 24 x 24mm</td><td>Knittel Glass</td><td>VD1 2424 Y100</td><td></td></tr><tr><td>Slides (Starfrost - ground edges 90)</td><td>Knittel Glass</td><td>VS1137# 077FKB</td><td></td></tr><tr><td>EDTA Tube</td><td>Greiner Bio-One</td><td>454034</td><td></td></tr><tr><td>Pasteur Pipette 150 mm (capillary tube)</td><td>Fisherbrand</td><td>1154-6963</td><td></td></tr><tr><td>26 G needle</td><td>Terumo</td><td>NN-2613R</td><td></td></tr><tr><td>&amp;nbsp;insulin syringe and needle 29 G</td><td>Terumo</td><td>BS05M2913</td><td></td></tr><tr><td>30 G needle&amp;nbsp;</td><td>Becton &amp;amp; Dickinson</td><td>304000</td><td></td></tr><tr><td>Flow cytometry tubes (blue)</td><td>Beckman Coulter</td><td>2523749</td><td></td></tr><tr><td>Water-repellent pen (Dakopen)</td><td>Dako</td><td>S200230</td><td></td></tr><tr><td>Sharp sterile scissors&amp;nbsp;</td><td>Nessi-care</td><td>SCI-01</td><td></td></tr><tr><td>Sterile forceps</td><td>Dominique Dutscher</td><td>956506</td><td></td></tr><tr><td>Thoma cell counting chamber</td><td>VWR</td><td>631-0397</td><td></td></tr><tr><td>Petri Dishes (Fisherbrand Plastic)</td><td>ThermoFisher Scientific</td><td>S33580A</td><td></td></tr><tr><td>Microcentrifuge tube (1.5 ml)</td><td>ThermoFisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Cylindrical Restrainer 15 - 30 gm</td><td>Stoelting</td><td>51338</td><td></td></tr><tr><td>Shandon Cytospin 3 Cytocentrifuge</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>10 ml syringe</td><td>Terumo</td><td>SS-10L</td><td></td></tr><tr><td>1 ml syringe</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>1.08543</td><td></td></tr><tr><td>Sterile gauze sponges</td><td>URGO</td><td>501580</td><td></td></tr></tbody></table>

a detailed protocol for characterizing the murine c1498 cell line and its associated leukemia mouse model

L&#39;iniezione endovenosa di C1498 cellule in topi singenici o congenic è stata eseguita dal 1941. Queste iniezioni provocare lo sviluppo di leucemia acuta. Tuttavia, la natura di questa malattia non è stato ben documentato in letteratura. Qui, forniamo un protocollo tecnico per caratterizzare cellule C1498 in vitro e per determinare la natura della leucemia indotta in vivo. La prima parte di questa procedura è focalizzata sulla determinazione del lineage ematopoietiche e la fase di differenziazione delle cellule C1498 coltivate. Per ottenere questo, multi-parametrica colorazione citometria di flusso è utilizzato per rilevare marcatori cellulari ematopoietiche. immunofluorescenza, citochimica e colorazione May-Grünwald Giemsa vengono eseguiti per valutare l&#39;espressione di mieloperossidasi, l&#39;attività di esterasi e morfologia cellulare, rispettivamente. La seconda parte di questo protocollo è dedicata a descrivere la malattia leucemia che è indotta invivo. Quest&#39;ultimo può essere ottenuto determinando le frequenze di cellule leucemiche e insiti nel sangue, organi ematopoietici (ad esempio, midollo osseo e milza) e tessuti non linfoidi (ad esempio il fegato e polmoni) utilizzando colorazione specifica e citometria a flusso analisi. La natura della leucemia viene poi confermata con May-Grünwald Giemsa colorazione e colorazione per esterasi specifiche nel midollo osseo. Qui, vi presentiamo i risultati che sono stati ottenuti utilizzando questo protocollo nei topi C1498- e PBS-iniettati di pari età.

A caratterizzare il modello C1498 del mouse, si è proceduto con due fasi principali. Innanzitutto, le cellule C1498 sono stati caratterizzati per determinare la loro ematopoietiche lineage e maturazione fase in vitro (Figura 1). Queste cellule sono state iniettate in topi congenic, e la natura della malattia leucemica indotta è stata valutata per determinare caratteristiche differenti: infiltrazione di cellule leucemiche, il loro fenotipo, la quantificazione delle cellule ematopoietiche (mature e progenitori / precursori) nel midollo osseo, le frequenze di C1498 cellule e cellule ematopoietiche mature nel sangue e una valutazione di gonfiore organo (nella milza, fegato e polmoni) e composizione cellulare. Per caratterizzare i fenotipi di cellule C1498 in vitro, le cellule sono state marcate con anticorpi diretti contro molecole che sono espresse da precursori emopoietici e cellule mature (Tabella 1), ei risultati sono stati analizzati utilizzando cytomet flussory. Le cellule C1498 sono stati positivi per l&#39;espressione superficie cellulare di Mac-1 (CD11b / CD18) (~ 7%), B220 (&gt; 25%), e sono visualizzati espressione intracellulare di CD3ε, T-cellule recettore catene (TCR) Vβ e Mac -3 (figure 2A e B). Le celle sono risultati negativi per i marcatori di superficie cellulare Ly6G, Ly6C, CD115, CD21 / CD35, CD19, CD3, CD4, CD8, NK1.1, e pan-NK molecole e per l&#39;espressione intracellulare di CD4 e CD8 (dati non riportati) . Sono stati poi esaminati per i marcatori di cellule staminali ematopoietiche e progenitori (Tabella 1). Essi sono stati anche negativi per l&#39;espressione della superficie delle cellule del CD117, CD34, Sca-1, CD150 e CD16 / 32 (dati non riportati). Queste cellule leucemiche sono stati poi sottoposti a test per determinare l&#39;espressione di adesione, presentazione dell&#39;antigene e molecole co-stimolatore. Le cellule espressi i marcatori di superficie LFA-1 (CD11a / CD18), CD44, CD31 (PECAM-1), e H-2D B e sono risultati negativi per MHC di classe II, CD80, CD86 e CD274 (dati non riportati). C1498 cellule Therefore espresso sia mieloide (Mac-1, Mac-3) e marcatori linfoidi (B220, CD3, TCR). Per caratterizzare meglio il loro lignaggio ematopoietico, espressione mieloperossidasi è stata valutata utilizzando la microscopia di immunofluorescenza. Tutte le cellule erano positive per la mieloperossidasi, che ha verificato la loro origine mieloide (Figura 3A). La maggior parte delle cellule anche macchiato positivo per esterasi α-naftil butirrato (Figura 3B, pannello di sinistra), e alcuni di loro macchiato per le naftolo AS-D cloroacetato esterasi (frecce nere) (Figura 3B, pannello di destra). I risultati indicano che le cellule contengono miscele di cellule monociti e granulocitari. Dopo il maggio-Grünwald Giemsa colorazione è stata eseguita, le cellule C1498 sono state osservate per visualizzare una morfologia blast-come con un elevato rapporto nucleo-citoplasmatica, da 3 a 5 nucleoli nel nucleo, un alone perinucleare, numerosi vacuoli e un citoplasma basofile (Figura 3C ). thnoi, la linea cellulare C1498 è composta da monoblasti e mieloblasti. Le cellule C1498 (CD45.2 +) sono stati poi iniettata per via endovenosa in CD45.1 + topi. I topi ha ceduto 17 a 19 giorni dopo le cellule sono state iniettate. Questi topi sono stati sacrificati in modo che il loro tipo di leucemia potrebbe essere analizzato prima di morire a causa della malattia. I topi di controllo, che sono stati iniettati con PBS, sono stati analizzati negli stessi punti di tempo per il confronto. I topi C1498 cellule iniettate visualizzata massiva infiltrazione di cellule C1498 nel loro midollo osseo, come dimostrato dalla comparsa blast-simili delle cellule dopo May-Grünwald Giemsa è stato eseguito (Figura 4A). Essi hanno inoltre conservato la loro monociti e fenotipi granulocitari (Figura 4B e C), dimostrando un accumulo di cellule monoblastiche e mieloblastiche che è caratteristica della leucemia mielomonocitica acuta. Da fareetermine se midollari numeri cellule ematopoietiche sono state inferiori a seguito dell&#39;invasione cellule leucemiche, CD45.2 + C1498 cellule, B linfocitica, monociti e le popolazioni granulocitari (compresi i progenitori, i precursori e cellule mature), sono stati quantificati con immunofluorescenza colorazione e il flusso multi-parametrica citometria. cellule leucemiche rappresentati da 16 a 36% delle cellule ematopoietiche (dati non mostrati). Gli altri tipi di cellule sono stati tutti presenti in numero significativamente più bassi nei topi C1498-iniettati che nei topi PBS-iniettati (per 5 volte, in media, per i sottoinsiemi di cellule B, media per le cellule granulocitari e 3 volte in media 4 volte su per sottoinsiemi monociti) (Figura 5A a C). Un&#39;indagine delle frequenze di cellule mononucleari in campioni di sangue leucemiche e mouse di controllo ha mostrato di contenere una percentuale comparabile di linfociti (Figura 6A), ma una maggiore frequenza di monociticae cellule leucemiche. Queste caratteristiche sono rappresentative della leucemia mielomonocitica acuta 11 (figura 6B). Tra le altre caratteristiche di leucemia mielomonocitica acuta 12, i topi C1498-iniettati presentati con fegati gonfi (epatomegalia), polmoni e milza (splenomegalia) (Figura 7a). Varie frequenze di cellule CD45.2 + C1498 sono stati rilevati in questi organi usando immunofluorescenza colorazione e citometria a flusso (Figura 7B). Come splenomegalia può derivare da un numero elevato di monociti infiltrati, abbiamo anche stimato le proporzioni delle popolazioni della milza. Il numero di cellule nel B linfocitica, monocitica e frazioni cellulari granulocitari erano significativamente più grande, con una media di 2 volte, 2,5 volte e 3 volte, rispettivamente, nella milza leucemiche che in milza controllo (Figura 7c). thin-page = &quot;1&quot;&gt; <img alt="Figura 1" src="/files/ftp_upload/54270/54270fig1.jpg" /> Figura 1. Rappresentazione schematica del protocollo Set Up per Caratterizzare in vitro coltivate C1498 linee cellulari e in descrizioni vivo di leucemia acuta. Il lignaggio ematopoietiche e la fase di differenziazione delle cellule C1498 tessuti in coltura sono stati prima determinata. cellule C1498 sono state iniettate in topi congenic per indurre lo sviluppo di leucemia acuta. L&#39;isolamento di midollo osseo, sangue periferico, milza, fegato e tessuti del polmone è stato effettuato per determinare le frequenze, fenotipi e cambiamenti morfologici dopo che le cellule infiltrazione C1498. IV: MGG endovenosa:. May-Grünwald Giemsa <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <html> annuncio / 54270 / 54270fig2.jpg &quot;/&gt; Figura 2. Analisi fenotipica delle cellule C1498 dopo coltura in vitro. Flusso rappresentante citometria dot plots e istogrammi di superficie delle cellule (A) e intracellulare (B) C1498-espresso molecole che sono stati associati con ematopoietiche differenziazione delle cellule mature sono mostrati. cellule C1498 sono state raccolte da culture, lavate ed etichettati utilizzando anticorpi fluorescenti che erano specifiche per la superficie delle cellule CD11b, CD18 e marcatori B220 o loro controlli isotipo. Per la colorazione intracellulare, le cellule sono state fissate, permeabilizzate ed etichettati utilizzando anticorpi diretti contro Mac-3, CD3ε, e un epitopo comune del TCR (T-Cell Receptor) catena Vβ o loro controlli isotipo. Le analisi sono state eseguite utilizzando gating con cellule vive. <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig2large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a> <html> S copi &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="Figura 3" src="/files/ftp_upload/54270/54270fig3.jpg" /> Figura 3. Caratterizzazione funzionale e morfologica delle cellule in coltura C1498. Cellule C1498 sono state raccolte da culture e centrifugate su vetrini per la microscopia. (A) di colorazione per l&#39;espressione mieloperossidasi è stata effettuata utilizzando immunofluorescenza. (B) reazioni citochimici sono stati usati per analizzare l&#39;esterasi α-naftil butirrato (NBE) e naftolo AS-D cloroacetato esterasi (CAE) attività in cellule C1498. Le cellule sono state considerati positivi per ciascuna etichetta quando marrone e rosso-viola, grandi granuli citoplasmatici, rispettivamente, sono stati osservati. (C) May-Grünwald Giemsa (MGG) colorazione C1498 cellule. Per ogni esperimento colorazione, l&#39;ingrandimento dell&#39;obiettivo microscopio è indicato. Ogni immagine rappresentativa tre esperimenti separati.large.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura. <img alt="Figura 4" src="/files/ftp_upload/54270/54270fig4.jpg" /> Figura 4. I topi di midollo osseo Morfologie in PBS- e C1498-iniettato. Cellule del midollo osseo sono stati isolati da PBS- e topi cellule iniettate C1498 e centrifugati su vetrini per la microscopia. (A) May-Grünwald Giemsa (MGG) colorazione. (B ) esterasi α-naftil butirrato (NBE) e naftolo (C) AS-D funzioni cloroacetato esterasi (CAE) sono stati valutati utilizzando citochimica. Nel pannello A, la band (immaturi) o segmentati neutrofili (maturi) sono meno visibili nel midollo osseo dei topi C1498-iniettati rispetto ai topi PBS-iniettati. Pannello B e C indicano che c&#39;era un accumulo di cellule monocitiche e granulocitari nel midollo osseo leucemico rispetto ai numeri osservati nel controllo midollo osseo. Tuttianalisi al microscopio sono state eseguite utilizzando un obiettivo di ingrandimento 100X. <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig4large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 5" src="/files/ftp_upload/54270/54270fig5.jpg" /> Figura 5. I topi analisi quantitativa delle popolazioni midollari in PBS- e C1498-iniettato. Cellule del midollo osseo sono stati isolati da PBS- e topi cellule iniettate C1498 e stimati dopo il conteggio delle cellule è stata eseguita. Le frequenze delle varie popolazioni cellulari sono stati determinati dopo immunostaining e il flusso delle cellule gated vivo analisi di citometria a. (A) I sottoinsiemi di cellule B inclusa + cellule CD19 + B220 a fasi, dalla cellule pro-B a maturare linfociti B (B), le cellule granulocitari nel CD3 - e CD11b + Ly6G + lignaggi, che comprendeva un precursori. ND granulociti immaturi e maturi (C) I sottoinsiemi monociti sono stati definiti come CD3 - CD115 + e inclusi cellule nel progenitrici di maturare fasi monociti. n = 7 topi / gruppo, ei dati sono presentati come istogrammi mostrano le medie ± SEM. ***, P &lt;0,0001 e **, p &lt;0,01, test di t-di Student spaiato confrontando PBS- e topi C1498-iniettati. <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig5large.jpg" target="_blank">Fai clic qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 6" src="/files/ftp_upload/54270/54270fig6.jpg" /> Figura 6. Analisi del sangue di mononucleari sottopopolazioni cellulari in PBS- e topi C1498-iniettato. Flusso rappresentante citometria punti appezzamenti di (A) T e B percentuali linfociti, che sono stati, rispettivamente, definiti come le cellule CD3 + + e B220 in PBS- e C1498 cellule iniettate. topi (B) frequenze di cella monocitica in C1498 leucemica e controllo (PBS) i topi sono stati determinati analizzando CD115 + Ly6C - e CD115 + Ly6C alte cellule. L&#39;analisi è stata eseguita da gating cellule vive. Per confrontare i topi leucemici e controllo, CD45.2 + sono stati esclusi C1498 cellule. <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig6large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 7" src="/files/ftp_upload/54270/54270fig7.jpg" /> Figura 7. Stima della splenici popolazioni leucemiche e topi di controllo. (A) fotografie rappresentativi di fegato, polmone e della milza gonfiore nei topi leucemica rispetto ai topi di controllo. Milze sono stati raccolti e pesati, e splenociti sono stati contati dopo interruzione del tessuto. (B) Istogramma che rappresenta le frequenze di cellule leucemiche in difforgani erenti dopo immunocolorazione è stata effettuata per le cellule CD45.2 + ei risultati sono stati analizzati mediante citometria a flusso. (C) stima della milza B, il numero di cellule granulocitari e monociti dopo immunostaining e citometria a flusso di analisi gating sono stati eseguiti per identificare vivo CD19 + B220 + , CD3 - CD11b + Ly6G +, CD3 - CD11b + Ly6C - e CD3 - alte cellule CD11b + Ly6C. Le barre di scala indicati per i polmoni, milza e fegato indicano 1 cm. .. n = 5 - 8 topi / gruppo, ed i dati sono rappresentati in istogrammi come mezzo ± SEM *, p &lt;0,05; **, p = 0,0033 topi, t-test di Student spaiato confrontando PBS- e C1498-iniettato <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig7large.jpg" target="_blank">prega clicca qui per vedere una versione più grande di questa figura.</a> <table fo:keep-together.within-page="1"><tbody&gt; <tr><td> Tipo di cella </td><td> Membrana o intracellulari Molecole </td></tr><tr><td></td><td></td></tr><tr><td> Precursori e cellule mature </td><td></td></tr><tr><td> Le cellule NK </td><td> NK1.1 +, pan-NK + </td></tr><tr><td> cellule NKT </td><td> NK1.1 +, pan-NK +, TCR vBeta + (8.2), CD3 + </td></tr><tr><td> linfociti T </td><td> TCR vBeta +, CD3 +, CD4 +, CD8 + </td></tr><tr><td> cellule B precursori e linfociti B </td><td> B220 +, CD19 +, CD21 / 35 + </td></tr><tr><td> precursori granulocitari e granulociti </td><td> Ly6G +, Mac-1 +, CD11b + </td></tr><tr><td> precursori monociti e monociti / macrofagi </td><td> CD11b +, Mac-1 +, Mac-3 +, CD21 / 35 +, CD115 +, Ly6Chi </td></tr><tr><td></td><td></td></tr><tr><td> progenitori </td><td></td></tr><tr><td> progenitori multipotenti </td><td> CD117 + Sca-1+ CD34 + (Lin- CD150-) </td></tr><tr><td> progenitori multipotenti linfoidi-innescato </td><td> CD117hi Sca-1hi CD127 + (Lin-) </td></tr><tr><td> progenitori linfoidi comuni </td><td> CD117lo Sca-1LO CD127 + (Lin-) </td></tr><tr><td> progenitori mieloidi comuni </td><td> CD16 / CD117 + 32lo CD34int (Lin- Sca-1-) </td></tr><tr><td> progenitori granulociti-macrofagi </td><td> CD16 / CD117 + 32hi CD34hi (Lin- Sca-1-) </td></tr><tr><td> progenitori megacariociti-eritroide </td><td> CD16 / CD117 + 32lo CD34lo (Lin- Sca-1-) </td></tr><tr><td></td><td></td></tr><tr><td> Le cellule staminali ematopoietiche </td><td> CD117 + Sca-1 + CD150 + (Lin- CD34-) </td></tr></tbody></table> Tabella 1. Marcatori di linee cellulari ematopoietiche e la differenziazione. CD: cluster di differenziazione; Lin: marcatori di cellule mature; Lo:bassa espressione; Max: elevata espressione; int: espressione intermedio; NK: cellule killer naturali; TCR: recettore delle cellule T.

Watch this Scientific Journal Video about A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model at JoVE.com

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

Questo manoscritto fornisce una procedura tecnica che può essere utilizzato per caratterizzare colture cellulari C1498 in vitro e la leucemia acuta indotta nei topi dopo l&#39;iniezione. Fenotipiche e funzionali analisi vengono eseguite utilizzando citometria a flusso, microscopia a immunofluorescenza, citochimica e maggio-Grünwald Giemsa.

Un protocollo dettagliato per caratterizzare la linea cellulare murina C1498 e il suo modello associato leucemia mouse

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

a-detailed-protocol-for-characterizing-murine-c1498-cell-line-its

Research

JoVE Journal

Cancer Research

19.9K Views.  INSERM, UMR-S-1172, Centre de Recherche Jean-Pierre Aubert (JPARC).  Questo manoscritto fornisce una procedura tecnica che può essere utilizzato per caratterizzare colture cellulari C1498 in vitro e la leucemia acuta indotta nei topi dopo l'iniezione. Fenotipiche e funzionali analisi vengono eseguite utilizzando citometria a flusso, microscopia a immunofluorescenza, citochimica e maggio-Grünwald Giemsa. 

Video: A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

Balmer Series

The Bohr Model
Niels Bohr proposed a model for the hydrogen atom in 1913 that described discrete energy states are associated with a fixed electron orbit around the nucleus. Importantly, an atom cannot discharge energy while its electrons are in stationary states. An electron can only emit energy by changing energy states. To change energy states, an electron must move from one orbit to another by either absorbing or emitting energy. This change can only occur if the absorbed or emitted energy equals the difference between the two states. Electrons cannot exist between orbits.
The quantum number, n, is used to label the different energy states. The lowest energy state is the ground state, which is n equal to one. The excited states are labeled n equal to 2, 3, 4, and so on. When an electron at the ground state absorbs a photon whose energy equals the difference between the ground and second state, the electron becomes excited and transitions from the ground state to the n= 2 excited state. If the energy of the photon equals the difference between the ground and third state, the electron moves to the n=3 state.
According to Bohr’s model, the potential energy of an electron in the nth level can be calculated using the following equation:
<img src="https://www.jove.com/CDNSource/lm/labs/54/54_Concepts_1.jpg" alt="Bohr model energy equation \(E_n=-\frac{Rhc}{n^2}\), quantum mechanics concept." data-altupdatedat="1747908711000">
where En is the potential energy, R is the Rydberg constant (1.0974 × 107 m-1), h is Planck's constant (6.62607004 × 10-34 m2·kg/s), and c is the speed of light (~ 3 × 108 m/s). Electrons can also spontaneously return to the ground state or any other lower excited state. When this happens, the excess energy is released in the form of an emitted photon. The energy of the photon is equal to the energy difference between the higher and lower energy states. That energy corresponds to wavelengths of light. Since each type of atom has different energy levels, the light emitted from each transition varies for each atom. For a sample of mixed molecules, the emitted light contains a range of wavelengths in what is called a continuous spectrum. For a sample containing atoms of a single element, the emitted light contains only certain wavelengths, which can be viewed as discrete lines once separated by a prism.
<h4>The Hydrogen Atom</h4>
Looking specifically at the hydrogen atom, the excitation of its electrons requires the absorption of sufficient energy to split the bond in the diatomic molecule H2. Since more energy is used to split the molecule than needed, the electrons in the hydrogen atom absorb the excess energy and are excited to a higher energy level. When the electrons spontaneously return to a lower energy level, light is emitted, which corresponds to the energy difference between the excited level and lower level.
When discussing the emission of energy, the higher energy level is considered the initial level, or ni, while the lower level is considered the final level, or nf. The wavelengths of emitted light ultimately depend on the energy difference between the two levels.
In a pure sample of hydrogen gas, the emission spectrum appears as distinct lines of discrete wavelengths that are specific to the element hydrogen. Some of these lines are in the visible range of the electromagnetic spectrum, while some are in the ultraviolet or infrared range.
<h4>The Balmer Series</h4>
The series of visible lines in the hydrogen atom spectrum are named the Balmer series. This series of spectral emission lines occur when the electron transitions from a high-energy level to the lower energy level of n=2. Johann Balmer observed these spectral lines at 410.2 nm, 434.1 nm, 486.1 nm, and 656.3 nm, which correspond to transitions from the n=6, n=5, n=4, and n=3 energy levels to the n=2 level, respectively.
Balmer was able to relate these wavelengths of emitted light using the Balmer formula.
<img src="https://www.jove.com/CDNSource/lm/labs/54/54_Concepts_2.jpg" alt="Wavelength formula λ=C(m²/(m²−n²)); physics equation; diagram for wave analysis." data-altupdatedat="1747908711000">
Here, λ is the observed wavelength, C is a constant (364.50682 nm), n is the lower energy level with a value of 2, and m is the higher energy level, which has a value greater than 3. This observation was then refined by Johannes Rydberg, where R is the Rydberg constant.
<img src="https://www.jove.com/CDNSource/lm/labs/54/54_Concepts_3.jpg" alt="Rydberg formula for spectral lines, equation, physics, spectroscopy calculation." data-altupdatedat="1747908711000">
Remember that this equation describes emitted light, so the higher energy level is considered the initial level, or ni, while the lower level is considered the final level, or nf. In the case of the Balmer series, nf is equal to 2. This equation was combined with the Bohr model to calculate the energy needed to move an electron between its initial and final energy levels, ΔE.
<img src="https://www.jove.com/CDNSource/lm/labs/54/54_Concepts_4.jpg" alt="Energy change formula, ΔE=Rhc(1/nf²-1/ni²), spectral transition equation, quantum physics." data-altupdatedat="1747908711000">
Later, other spectral series for the hydrogen atom were discovered. For example, the Lyman series contains emission lines with energies in the ultraviolet region.
<h4>References</h4>
<ol>
	<li style="font-size:17px">Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2012). Chemistry and Chemical Reactivity. Belmont, CA: Brooks/Cole, Cengage Learning.</li>
	<li style="font-size:17px">Silderberg, M.S. (2009). Chemistry: The Molecular Nature of Matter and Change. Boston, MA: McGraw Hill.</li>
</ol>

The Hydrogen Atom: Bohr Model and Balmer Series - Concept

Serie Balmer

The Bohr Model
Niels Bohr proposed a model for the hydrogen atom in 1913 that described discrete energy states are associated with a fixed electron orbit ...

Didattica

JoVE Lab Manual

Chimica

Genetics of Organisms

<h4>Mendelian Genetics</h4>
Evolution is caused by changes in the genetic composition of populations. In the field of population genetics, scientists model this process as changes in the frequency of alleles at individual genetic loci. This simple representation of how evolution occurs dates to Gregor Mendel&#8217;s analysis of trait inheritance patterns in pea plants, first presented in 1865. Mendel determined, using rigorous collection of data, that noticeable traits are controlled by two alleles of each gene (he called them factors), one from each parent. In addition, he concluded that some alleles are dominant to others (deemed recessive). Interestingly, Mendel&#8217;s data was not appreciated until 1900, probably in part because his approach to the study of inheritance was so divergent from his predecessors. Even after his brilliant experiments were rediscovered, it took 30 additional years for Mendelian inheritance to become integrated with evolutionary theory and to gain universal acceptance.
Why was such a seemingly clear and straightforward process dormant for so long and then the center of controversy for decades? One key to the controversy is that we rarely see the clear patterns of inheritance described by Mendel. Animal and plant breeders most often see evidence for what was described as blending inheritance, or offspring whose phenotypes are intermediate between those of their parents, rather than the discrete segregation of traits into well-defined categories, like smooth versus wrinkled peas or red versus white flowers. Mendelian inheritance thus did not align well with practical experience and it took decades to reconcile the two. Additionally, there are now known to be modes of inheritance that, at first glance, appear to violate Mendel&#8217;s laws. Deeper investigations into genetics using molecular biology have effectively characterized these novel modes of inheritance. The new information does not invalidate Mendel&#8217;s findings, however, it simply enhances his model.
<h4>Phenomena that Violate Mendelian Rules</h4>
There are three main phenomena: epistasis, pleiotropy, and sex-linkage that appear to violate the basic dominance and recessivity rules of Mendelian inheritance. Epistasis occurs when two or more genes or alleles interact to affect a phenotype. A function of one gene product may be required to allow another gene to be expressed or to function normally. On the other hand, pleiotropy occurs when one gene controls the expression of multiple phenotypes in an individual. For example, a large proportion of cats with white fur and blue eyes are deaf 1. Pleiotropy is often antagonistic, meaning that the same gene causes beneficial changes in one facet of an individual&#8217;s phenotype while at the same time causing damaging changes in some other aspect of an individual&#8217;s phenotype. Antagonistic pleiotropy is seen as a tradeoff or constraint on evolution, as proposed by George C. Williams. For example, the age specific decline in performance, or senescence, is thought to be a pleiotropic trait controlled by the same genes that increase early-life fecundity2,3.
<h4>Sex Linkage</h4>
Sex linkage is another phenomenon discovered as a counter-example to basic Mendelian inheritance. In many organisms, pairs of sex chromosomes determine gender. In humans and many mammals, XX individuals are female while XY individuals are male. Male offspring inherit an X from their mother and a Y from their father; females inherit an X from their mother and an X from their father. In most species, the ratio of males to females at birth under &#8220;normal&#8221; environmental conditions is 1:1. Y-chromosomes undergo little recombination with X chromosomes during meiosis, due to the fact that, over evolutionary time, they have lost a substantial number of genes. Interestingly, there are only 16 genes shared by the human X and Y chromosomes4.
For X-linked traits (traits encoded by genes on the X chromosome) females can be homozygous (having two copies of the same allele) or heterozygous (having two different alleles). If the trait is recessive, females will only show the phenotype for the trait if they are homozygous. Heterozygous females &#8220;carry&#8221; the recessive allele, but do not express the phenotype of that allele. Statistically speaking, half of her sons will inherit this recessive allele and all will express the phenotype because males have only one X chromosome.
A commonly used example of an X-linked phenotype is hemophilia, a disease that prevents the synthesis of blood clotting proteins. If a female is heterozygous (is a carrier, genotype XH Xh) mates with a normal male (genotype XH Y), 50% of the sons will be affected. If the female was instead homozygous for the trait (genotype Xh Xh ), none of her daughters would be hemophiliacs but 100% of her sons would be. The differential phenotypic ratios seen in sons and daughters appears to violate Mendel&#8217;s laws, yet Mendel&#8217;s first law still applies; each offspring has a 50% chance of inheriting an X-chromosome carrying the hemophilia allele from a heterozygous mother5.
Like humans, the fruit fly Drosophila melanogaster has an XY-sex determination system. D. melanogaster make excellent laboratory organisms because they are easy to keep, breed, and manipulate. Wild-type flies have what is considered normal body morphology and red eyes. Many lines are readily available for purchase, including some with mutations resulting in flies without eyes, with eyes of varying color, or missing wings. These organisms can be used to study or demonstrate both Mendelian and non-Mendelian inheritance patterns. In fact, 1933 Nobel Prize winner, Thomas Hunt Morgan, first noticed that some genes are sex-linked using this system6.
<h4>References</h4>
<ol class="references">
	<li>Strain, George M., &#39; Cat Breeds With Congenital Deafness,&#39; [Online]. Available: https://www.lsu.edu/deafness/catbreeds.htm. [Accessed 29 August 2018].</li>
	<li>W. GC., &#39;Pleiotropy, natural selection, and the evolution of senescence.,&#39; Evolution, vol. 11, p. 398&#8211;411, 1957.</li>
	<li>T. &#38; S. P. S. Flatt, &#39;Integrating Evolutionary and Molecular Genetics of Aging,&#39; Biochimica et Biophysica Acta, vol. 1790, no. 10, p. 951&#8211;962, 2009.</li>
	<li>D. Bachtrog, &#39;Y chromosome evolution: emerging insights into processes of Y chromosome degeneration,&#39; Nature Reviews. Genetics, Vols. 14(2), 113&#8211;124, 2013.</li>
	<li>US National Library of Medicine, &#39;Hemophilia,&#39; Genetics Home Reference, [Online]. Available: https://ghr.nlm.nih.gov/condition/hemophilia. [Accessed 31 August 2018].</li>
	<li>&#39;The Nobel Prize in Physiology or Medicine 1933,&#39; [Online]. Available: https://www.nobelprize.org/prizes/medicine/1933/summary/. [Accessed 31 August 2018].</li>
</ol>

Genetics of Organisms: REF and WEF Fly Genetics - Concept

Genetica degli organismi

Mendelian Genetics
Evolution is caused by changes in the genetic composition of populations. In the field of population genetics, scientists model this ...

Biologia

Genetica

Animal Behavior

<h4>Drosophila as a Model Organism</h4>
The common fruit fly, Drosophila melanogaster, is a widely used model organism in biology, though it may be more commonly recognized as a fruit-loving pest. There are multiple reasons that make D. melanogaster an excellent experimental organism: their roughly two-week generation time allows studying multiple generations, they are easily maintained in very small tubes, and their sexual dimorphism enables investigators to easily distinguish between males that have black coloration towards the posterior and females that have banded abdomens.
Thomas Hunt Morgan and his students were among the first to make use of Drosophila, in the early 20th century. In 1933, Morgan received a Nobel Prize for his discovery of the role of chromosomes in heredity, which was founded on his observations of mutant Drosophila. Morgan continued his studies by generating new mutants with radiation. The presence of multiple mutants with discrete effects on the appearance of the fly enabled him to look at patterns of multifactor inheritance. A key aspect of his early work was the discovery of apparent violations of Mendel&#8217;s second law, which states that genes segregate independently of one another. Morgan&#8217;s observations indicated that genes will not segregate independently if they are joined together on the same chromosome. Furthermore, it became possible to determine gene locations on a chromosome relative to each other through analyses of how often they recombined thus triggering gene mapping studies to identify the location of a gene and distances between genes. Consequently, associates of Morgan&#8217;s lab received two additional Nobel Prizes for other studies of Drosophila genetics. Morgan&#8217;s success and the accumulating information about Drosophila inspired others to adopt them as experimental organisms, which added to our knowledge of them and progressively increased their value as a model organism. Other scientists have developed many mutations. Over 27,000 unique Drosophila lines are now maintained as stock cultures at the Drosophila stock centers and are readily available to laboratories that use them as tools for studying diverse aspects of biology, including behavior1.
<h4>Drosophila Ethology</h4>
Animal behavior, or ethology, is the study of how animals interact with each other and their environments. Although it is a relatively young biological field, ethology has important implications since an organism&#8217;s survival depends on how it behaves with regard to possible mates, food sources, and natural obstacles. Studying these organisms helps scientists to understand the contexts of these behaviors and the underlying mechanisms as to how they have evolved. Thus, a goal of the study of animal behavior is to find a genetic basis for the behaviors exhibited by individuals or groups of individuals. For example, kin selection, first proposed by William Hamilton, suggests that individuals will behave altruistically when making a sacrifice for a family member rather than a stranger2. This is because related individuals share more genes than unrelated ones, and the future reproduction by their relatives contributes in a way to their own fitness, or reproductive success. Hence, observing how organisms like Drosophila interact with their environment can allow us to understand how these behaviors are hard-wired. Additionally, these behaviors provide insight into how these organisms have adapted to cope with their environment and survive.
Directional behaviors are commonly studied to understand the basis of how and why an organism moves in its environment. Kinesis and taxis are two forms of directional behaviors. Kinesis involves stimulus-induced movements in random directions, such as the random movement of flies when chased away. On the other hand, taxis involves stimulus-induced movement towards a specific direction, such as moths flying towards a lightbulb. Taxis behaviors are positive if the animal moves towards the stimulus and negative if the animal moves away from the stimulus. Furthermore, taxis behaviors are named based on the specific stimulus that induces them. For example, geotaxis is a response to gravity, where positive geotaxis means that the organism moves with gravity. Phototaxis is movement in response to light, such as positive phototaxis of moths towards a lightbulb. Similarly, chemotaxis is attraction to or avoidance of an airborne chemical cue, such as the negative chemotaxis of many predators, including humans, in response to the smell of skunk spray. Furthermore, there are many other types of taxes that are studied by scientists, including aerotaxis, barotaxis, hydrotaxis, and magnetotaxis, that are movements in response to oxygen, pressure, water, and magnetic fields, respectively.
<h4>Study of Drosophila taxis behavior</h4>
A common and simple method to study Drosophila taxis behavior is to use a choice chamber, which allows the observation of a directional response of the fly to a particular stimulus, such as gravity, light, or a chemical. Drosophila are inserted in the center of the choice chamber and then allowed to wander freely about the chamber, which is designed so that alternative stimuli can be presented at opposing sides. After a period of wandering, the number of flies is counted on either side of the choice chamber. The number of flies on each side of the chamber is then analyzed with the Chi Square test to gauge whether or not there is a preference for a stimulus.
Comparison of directional behavior of mutant and wild-type Drosophila allows researchers to determine the role of the mutated gene in that specific behavior. Moreover, scientists are also trying to understand how animals decide what to do in the presence of combinations of stimuli, including conflicting stimuli3. Together, these studies can help predict how organisms will react to certain stimuli or changes in environmental factors.
<h4>References</h4>
<ol class="references">
	<li>Cook KR, Parks AL, Jacobus LM, Kaufman TC, Matthews K. New research resources at the Bloomington Drosophila Stock Center. Fly. 2010, Vol. 4, 1: 88-91.</li>
	<li>WD, Hamilton. Altruism and related phenomena, mainly in social insects. Ann Rev Ecol Systematics. 1972, Vol. 3, 193-202.</li>
	<li>Gepner R, Skanata MM, Bernat NM, Kaplow M, Gershow M. Computations underlying Drosophila photo-taxis, odor-taxis, and multi-sensory integration. eLife. 2015, Vol. 4, e06229.</li>
</ol>

Animal Behavior: Studying Taxis Behavior of Drosophila - Concept

Comportamento animale

Drosophila as a Model Organism
The common fruit fly, Drosophila melanogaster, is a widely used model organism in biology, though it may be more commonly ...

Ecologia

Hardy-Weinberg and Genetic Drift

Evolutionary change is interesting and important to study, but changes in populations occur over long periods of time and in huge physical spaces and are therefore very difficult to measure. In general, studying phenomena like this requires the use of mathematical models which are built using parameters that can be conveniently measured. These models are then used to make predictions about how changes to the system might affect the outcome. 
For example, changes to the frequency of genetic alleles at individual loci in species are often observed over long time periods, but are not generally observable over short time periods. The use of computer models allows researchers to predict changes within a species&#8217; gene pool using observations that have already been gathered, and allow for simulations of a potentially unlimited number of generations of the population. This would certainly not be possible within a human lifetime.
<h4>The Hardy-Weinberg Equilibrium Principle</h4>
One equation used to model populations is the Hardy-Weinberg equilibrium equation. It was formulated independently in 1908 by both G. H. Hardy and Wilhelm Weinberg1,2. The simple equation describes the expected allele frequency of a population that is not evolving. Because most real-life populations are evolving in response to the forces of natural selection, this formula acts as a useful null hypothesis. It is used to test whether or not different types of selection are, or are not, occurring. If measured allele frequencies differ from those that were predicted using the Hardy-Weinberg equation, then the gene in question is undergoing evolutionary change.
The Hardy-Weinberg equation can be represented as p2 + 2pq + q2 = 1 where p represents the frequency of the dominant allele and q represents the frequency of the recessive allele for the gene in question. Each expression in the equation represents the predicted frequency of one of the three possible diploid genotypes. Homozygous dominant frequency is represented by p2, homozygous recessive by q2, and heterozygous by 2pq. If these frequencies are summed, it adds up to 1, which makes sense since the full population had been divided into the three available categories.
It is also possible to describe the population, or gene pool, in terms of the two alleles, without regard to how they are packaged into diploid individuals. This equation is represented as p + q = 1, that is, the frequencies of the dominant and recessive alleles must add up to 1 within the population. Again, this makes sense, since the model includes just two allele possibilities. Notice that the frequency of genotypes in the population is just the quadratic expansion of the frequency of alleles in the population because (p + q)2 = p2 + 2pq + q2.
The Hardy-Weinberg equation, like most other models, requires a series of assumptions. Usefully, if the real-life data differs from the predicted data, it is possible to hypothesize which of the starting assumptions was false. The assumptions of the Hardy-Weinberg equilibrium equations are: 1) the population is very large, 2) the population is closed, meaning that there are no individuals immigrating into or emigrating out of the population, 3) there are no mutations occurring on the gene in question, 4) individuals within the population are mating at random&#8212;individuals are not choosing their mates, 5) natural selection is not occurring. Again, keep in mind that the Hardy-Weinberg equilibrium equation is a null hypothesis. Frequently, real populations violate one or more of these assumptions. The equation is used to determine if and how evolution is happening.
<h4>Genetic Drift</h4>
In a relatively small population, a condition that violates the first Hardy-Weinberg assumption, it is possible for allele frequencies to have resulted from chance. This phenomenon is referred to as genetic drift. One version of this is referred to as the founder effect. If a small number of individuals move to an isolated location and start a new population, the specific genetics of those particular individuals will shape the future generations. This new small gene pool may have the same allele frequency as the original, but it is also possible, even likely, that it does not. Say the original population included 50% dominant alleles and 50% recessive. In the extreme, if all of the individuals in a migrating founder group are homozygous recessive, then the dominant allele has been lost completely and the recessive is now at 100%. A similar phenomenon, called the bottleneck effect, can occur if a population sustains a large drop in numbers due to a natural disaster, human intervention, or disease.
Importantly, genetic drift represents a type of evolution that is not necessarily adaptive. It does not specifically select for traits that are fit for the environment. Nonetheless, it is an important evolutionary force in shaping populations with a small number of individuals and will cause deviations from Hardy-Weinberg predictions. In populations that are larger, deviations from the predictions are more likely to mean that the population in question is undergoing evolution through natural selection. Under these circumstances, further consideration of the mechanisms causing this evolution can be explored.
Modern applications of the Hardy-Weinberg model include analysis of human and animal populations in terms of how their immune systems relate to their susceptibility to infectious disease3,4. Many research groups are cataloging genes which encode specific immune system molecules, such as CCR5 and the major histocompatibility complex (MHC, known in humans as the human leukocyte antigen (HLA), and correlating this information to epidemiological studies regarding disease and its progression3,5-6. From comparing these two types of data sets, patterns are emerging showing that some individuals are genetically resistant to particular infections while others are more likely to succumb to disease4. Hardy and Weinberg&#8217;s model has been essential to the analysis of this data and has contributed to our understanding of how infections have shaped evolution.
<h4>References</h4>
<ol class="references">
	<li>Hardy, G. H. (Jul 1908). 'Mendelian Proportions in a Mixed Population' (PDF). Science. 28 (706): 49&#8211;50. doi:10.1126/science.28.706.49. ISSN 0036-8075. PMID 17779291.</li>
	<li>Weinberg, W. (1908). '&#220;ber den Nachweis der Vererbung beim Menschen'. Jahreshefte des Vereins f&#252;r vaterl&#228;ndische Naturkunde in W&#252;rttemberg. 64: 368&#8211;382.</li>
	<li>Eguchi S, Matsuura M. Testing the Hardy-Weinberg equilibrium in the HLA system. Biometrics. 1990 Jun;46(2):415-26. PubMed PMID: 2364131.</li>
	<li>Phillips KP, Cable J, Mohammed RS, et al. Immunogenetic novelty confers a selective advantage in host-pathogen coevolution. Proc Natl Acad Sci U S A. 2018;115(7):1552-1557.</li>
	<li>Lim JK, Glass WG, McDermott DH, Murphy PM CCR5: no longer a 'good for nothing' gene--chemokine control of West Nile virus infection. Trends Immunol. 2006 Jul; 27(7):308-12.</li>
	<li><a target="__blank" href="https://hla-net.eu/tools/frequency-estimation/">https://hla-net.eu/tools/frequency-estimation/</a></li>
	<li>Santiago Rodriguez, Tom R. Gaunt and Ian N. M. Day. Hardy-Weinberg Equilibrium Testing of Biological Ascertainment for Mendelian Randomization Studies. American Journal of Epidemiology Advance Access published on January 6, 2009, DOI 10.1093/aje/kwn359. http://www.oege.org/software/hwe-mr-calc.html</li>
</ol>
<h4>Further Reading</h4>
Andrews, C. (2010) The Hardy-Weinberg Principle. Nature Education Knowledge 3(10):65 https://www.nature.com/scitable/knowledge/library/the-hardy-weinberg-principle-13235724

Hardy-Weinberg Equilibrium Principle and Genetic Drift - Concept

Hardy-Weinberg e la deriva genetica

Evolutionary change is interesting and important to study, but changes in populations occur over long periods of time and in huge physical spaces and are ...

Evoluzione

Evolutionary Relationships

Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4th century BC. Aristotle&#8217;s system was improved upon during the Renaissance and then, subsequently, by Carolus Linnaeus in the mid 1700&#8217;s. These more formal classification and organization systems grouped species by their physical similarity to one another. For example, all vertebrates have a backbone, but invertebrates do not. Traits like the backbone are called synapomorphies, which are traits that are shared by a group of organisms, presumably because they were derived from a common ancestor. As we will explore, this method has been shown to have limitations and has more recently been amended to include genetic analysis. Still, scientists construct trees called dendrograms to create a visual representation of how species are related to one another and share common ancestors. These dendrograms can aid in our understanding of the evolutionary processes that drive these relationships. Genetic comparisons have added an important tool guiding the analysis of evolutionary relationships.
<h4>Types of Dendrogram</h4>
A type of dendrogram, called a cladogram, depicts the hypothetical genealogical relationships between species with the tips (or leaves) of the tree representing a species and the branches showing how species are related to each other. A slightly more complicated type of tree, called a phylogram, differs from a cladogram in that the branches leading to the species are of different lengths. The length of a branch in this type of tree represents the degree of change between species: the longer the branch, the more time since the species have diverged from a common ancestor. In both types of tree, the common ancestor of a group of species is indicated by a node, which is the point where a series of branches meet. Species that are more closely related to each other (most recently shared a common ancestor) will be located closest to the node. The two species that share a node are called a sister group1.
<h4>Understanding Evolutionary Relationships using Genetic Data</h4>
Historically, cladograms were constructed by comparing the morphology (physical structure) of organisms. This method is still practiced but the techniques have been modernized to include comparison of DNA (deoxyribonucleic acid) sequences between species. Using DNA for building trees has several advantages over relying solely on morphology, including being able to calculate an estimate of how long ago different species shared a common ancestor1. However, using DNA is not always feasible, especially when trees include extinct organisms. DNA is best found in soft tissues, which are not preserved during the fossilization process, and therefore it is uncommon for a DNA sample of an extinct species to be available.
DNA is passed on from parents to their offspring in hereditary units called genes. The nucleotide (A, G, C, and T) sequence of genes found in different species are frequently quite similar, likely due to their having come from a common ancestor. This fact allows researchers to align sequences from different species with one another to build the trees described above. Species with more similarity between their nucleotide sequences will be placed next to each other in a tree, and species with less sequence similarity will be placed further apart from each other.
Bioinformatics are the tools used by biologists to analyze large datasets using a combination of computer science, mathematical modeling, and statistics. One such tool is called BLAST (Basic Local Alignment Search Tool), which can be used to quickly search the entire genome of any species that is available in the NCBI (National Center for Biotechnology Information) database2. The NCBI database combines several different databases that hold different types of DNA sequence information. The process of a BLAST search includes complex computer algorithms, but basically, BLAST aligns sequences of each nucleotide base from a submitted DNA sequence (known as the query sequence) with sequences in the data base that most closely match it. The DNA sequences that are found will be listed in order of similarity to the sequence in question, and will therefore be from species closely related to the species containing the query gene. This comparison may or may not depict the actual evolutionary relationship between species because genes evolve at different rates. Additionally, genomes sometimes contain more than one instance of a similar sequence.
Comparison of DNA sequences of genes is valuable beyond consideration of evolutionary relationships. Frequently, genes are identified in model organisms, such as the fruit fly, Drosophila melanogaster, or the mouse3. Integral to studying a gene, the function of its product is commonly identified and analyzed. If a researcher is interested in studying that function in a different organism (humans for example), BLAST or other bioinformatic tools can be used to find candidate genes based on their similarities to the genes of known function from model organisms.
Human genes can also be used as the starting point to find homologs in model organisms. In fact, human disease research depends heavily on this. Once a human gene of interest is identified, mice can be genetically manipulated to have the homologous gene disrupted, or &#8220;knocked out,&#8221; creating a model of the human disease that can be studied in order to understand and treat the disease. There are many of these mouse strains currently available. For example, there is a mouse model for human Cystic Fibrosis (CF) called the Cftr knockout mouse and another modeling atherosclerosis, called the Apoe knockout3. 
 
<h4>References:</h4>
<ol class="references">
	<li>Annenberg Learner. Online Textbook: Unit 3 Evolution and Phylogenetics. Rediscovering Biology. [Online] 2017. [Cited: August 23, 2018.] https://www.learner.org/courses/biology/textbook/compev/compev_3.html.</li>
	<li>National Center for Biotechnology Information, U.S. National Library of Medicine. BLAST: Basic Local Alignment Search Tool. [Online] https://blast.ncbi.nlm.nih.gov/Blast.cgi.</li>
	<li>National Human Genome Research Institute. Background on Mouse as a Model Organism. National Human Genome Research Institute. [Online] December 2002. [Cited: August 23, 2018.] <a href="https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/" target="__blank">https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/</a>.</li>
</ol>

Evolutionary Relationships: Using BLAST to Test Evolutionary Hypotheses - Concept

Relazioni evolutive

Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4th century BC. Aristotle&#8217;s system ...

Animal Diversity

Kingdom Animalia is composed of a range of organisms united by a set of common characteristics. Barring a few exceptions, animals are multicellular eukaryotes that move, consume organic matter, and reproduce sexually. Although these attributes are shared, species within this kingdom are also extremely diverse. This diversity is due to adaptation of each species to a different niche. The niche of a species includes the area, function, and interrelationship of that species with other biotic and abiotic factors in its environment. Niche specialization through evolutionary adaptation allows species to survive and reproduce effectively in their environment and reduces competition among species within the same habitat1-2.
<h4>Classification</h4>
Diversity in Kingdom Animalia has led to the classification of 36 distinct phyla, based on their evolutionary lineage. Seven of these phyla will be discussed, including Porifera, Cnidaria, Platyhelminthes, Annelida, Mollusca, Arthropoda, and Chordata. The first, phylum Porifera, is the earliest, simplest, most ancestral phylum. It includes multicellular, asymmetrical filter feeding sponges that lack distinct tissue layers. Interestingly, sponges can regrow after being broken apart to the level of a single cell. Next, increasing in complexity, the phylum Cnidaria includes jellyfish and coral. Members of this phylum are radially symmetric and diploblastic (possessing two tissue layers). These tissue layers are called the endoderm, which makes up the inner layer, and the ectoderm, which forms the outer layer. Organisms in phylum Cnidaria contain a simple gut with a single opening through which food enters and waste is excreted. Both Porifera and Cnidaria include organisms that are largely sedentary as adults. However, the remaining phyla have evolved adaptations that allow movement and more sophisticated behavior.
Following Cnidaria, there are two phyla of worms, termed Platyhelminthes and Annelida. Worms from both of these phyla breathe by exchanging gases across their skin. As a result, most worms have a large surface area to volume ratio and live in moist or aquatic environments. The skin of these organisms must remain moist to facilitate effective diffusion of gases. Another common characteristic of worms is their bilateral symmetry. This means that an organism can be symmetrically divided in half along a plane. Bilateral symmetry is seen not only in worms, but also in every phyla from this evolutionary point forward. Such symmetry is important to allow directed movement and to concentrate sensory organs near the anterior tip, or head, where they interact with their surroundings. Worms and later phyla also contain an additional tissue layer called the mesoderm that lies between the ectoderm and endoderm. As a result, these organisms are considered &#8220;triploblastic.&#8221;
Of the two phyla of worms, Platyhelminthes is the more ancient and simple. These include flatworms and trematodes, which may be free-living or parasitic. Like Cnidarians, they possess a simple gut with a single opening. Phylum Annelida is more complex, including earthworms, leaches, and polychaeta. These organisms contain a body cavity, called the coelom, between the gut and body wall. They are also distinct due to the segmentation of their bodies. Division of an organism into segments was an important evolutionary step, allowing adaptation of each segment to specialized forms and functions. Major evolutionary advances in this phyla include the development of anterior segments with appendages specialized for grasping and processing food as well as posterior segments with appendages specialized for locomotion.
The next phylum, Mollusca, contains highly morphologically diverse organisms, including octopuses, snails, slugs, and clams. There are few defining features for this group. However, all mollusks possess a mantle, which acts as a protective cover for the respiratory organs, digestive tract, and reproductive structures. Next, the most diverse group of organisms is phylum Arthropoda. This phyla alone makes up approximately three quarters of all living and extinct species known today, including crustaceans, insects, and arachnids. All members of this phylum have a hard, outer coating made of chitin, called an exoskeleton. This exoskeleton is periodically shed for a new, larger one to take its place. Like Annelida, arthropods are segmented. However, instead of multiple repeating sections, arthropods are broken up into three distinct segments called the head, thorax, and abdomen. Most have specialized sensing structures attached to the head called antennae.
Lastly, phylum Chordata includes all animals with a backbone and a few without. Two defining features that unite this group are the presence of a nerve chord and a notochord. The nerve cord is a major part of the chordate nervous system and the notochord is a cartilaginous structure that runs between the nerve cord and the digestive tract. Phylum Chordata includes tunicates (which lose their nerve cord and notochord after metamorphosis from the larval to adult life stage), fish, amphibians, birds, reptiles, and mammals (including humans)3.
<h4>Form and Function</h4>
The vast diversity in body structure, habitats, and behavior observed across all 36 phyla within Kingdom Animalia is truly astounding. To understand these differences better, it is important to consider the connection between form and function. &#8220;Form&#8221; simply means the shape, size, and substance of a structure or organism while &#8220;function&#8221; stands for the way in which that structure is used. Accordingly, the function of a structure can be predicted based on the form observed, or vice versa. For example, a bird specialized to crack hard nuts would be predicted to have a sturdy, sharp beak while a bird that eats fruit may have a smaller, more rounded beak.
Importantly, the form of an organism is constrained by its physiological requirements. For instance, all animals must consume and digest organic matter and exchange gas with their environment. As a result, any adaptations to an animal&#8217;s form must continue to facilitate these processes efficiently. For example, in smaller animals, diffusion of gases across the skin is possible and often useful. However, as the size of an animal increases, its surface area to volume ratio decreases until diffusion across the skin can no longer provide sufficient gas exchange. Such larger animals have developed complex systems to deal with gas exchange, such as the lungs of a whale or the gills of a fish. As a result, the morphology of gas exchange systems depends on both the size of an organism and its environment.
Similarly, it generally follows with other types of adaptations that form follows function, constrained only by the physiological requirement of the organism. However, there are cases in which form does not follow function. Vestigial structures are those that served a purpose in an organism&#8217;s ancestor but are no longer useful in their current form. Such structures have little to no impact on the fitness of an individual, and are thus not evolutionarily selected for or against within the population. As a result, some structures persist with limited functional capacity. For example, the human appendix, a small pocket at the end of the large intestine, is an evolutionary remnant from our ancestors. While this structure has been hypothesized to store beneficial gut bacteria, removal of the appendix is possible with no deleterious effects.
<h4>Convergent Evolution</h4>
Among adaptations seen in Kingdom Animalia, many have developed multiple times throughout evolutionary history. Consider the wings of a bat and those of a bird as an example. These structures are not a result of evolutionary relatedness, but have instead arisen from convergent evolution4. Convergent evolution occurs when two organisms independently evolve similar traits due to similar environmental pressures or niches. The wings of bats, birds, and insets have all developed independently and are said to be &#8220;convergent traits.&#8221; Clearly, such traits are quite beneficial for the niches in which they arise.
<h4>References</h4>
<ol class="references">
	<li>Hollins, J., et al. (2018). 'A physiological perspective on fisheries-induced evolution.' Evol Appl 11(5): 561-576.</li>
	<li>Leong, M., et al. (2017). 'The Habitats Humans Provide: Factors affecting the diversity and composition of arthropods in houses.' Sci Rep 7(1): 15347.</li>
	<li>Wray, G. A. (2015). 'Molecular clocks and the early evolution of metazoan nervous systems.' Philos Trans R Soc Lond B Biol Sci 370(1684).</li>
	<li>Foll, M., et al. (2014). 'Widespread signals of convergent adaptation to high altitude in Asia and America.' Am J Hum Genet 95(4): 394-407.</li>
</ol>

Animal Diversity: Form and Function of Crickets and Crayfish - Concept

Diversità animale

Kingdom Animalia is composed of a range of organisms united by a set of common characteristics. Barring a few exceptions, animals are multicellular ...

Microbial and Fungal Diversity

Bacteria and fungi are two highly diverse groups of organisms that can have significant beneficial or detrimental impacts on human health. For this reason, it is important to understand and distinguish between individual species of these groups. As you will recall, biological taxonomists group organisms based on their phylogenetic relatedness. The three domains of life, Bacteria, Archaea, and Eukaryota segregate life into three distinct groupings. While all bacteria fall within the domain Bacteria, fungi are found in the domain Eukaryota (Kingdom Fungi). In addition to Archaea, domain Bacteria exclusively includes single-celled prokaryotes, meaning their cells lack both a nuclear membrane and membrane-bound organelles. In contrast, fungi of domain Eukaryota have membrane-bound organelles and include both unicellular and multicellular organisms.
<h4>Bacteria</h4>
Bacteria are found nearly everywhere on Earth and are highly adapted to the environments in which they grow. As a result, this domain includes a staggering amount of diversity. The impact of these organisms on human health is equally diverse. For example, species like Streptococcus thermophilus can be used to produce fermented foods like yogurt, while spores of the bacteria Bacillus anthracis are responsible for the potentially deadly disease anthrax.
To aid in the identification of such organism, microbiologist have developed a myriad of tests to characterize both individual cells and entire colonies of bacteria1. One of the most common methods for identifying bacteria is by Gram staining. This test uses colored dyes to determine the composition of the bacteria&#8217;s cell wall. Gram-positive bacteria, which bear a thick cell wall made up of a protein called peptidoglycan, retain a dye called crystal violet and thus appear purple. In contrast, gram-negative bacteria have a thinner cell wall without peptidoglycan and do not retain the crystal violet. Instead, these cells appear pink due to a counter stain called safranin. The use of Gram staining often serves as a preliminary step in the identification of a bacterial species. Further classification requires the identification of additional characteristics. This can include the shape of individual bacteria, which may be either cocci (round), spirillum (spiral), or bacilli (rod-shaped). Additionally, the color, shape, sheen, texture, and even smell of bacterial colonies can be used to distinguish between species.
<h4>Fungi</h4>
Fungi of domain Eukaryota are also highly ecologically diverse and can range in size from microscopic yeast to the largest organism on earth, the honey fungus (Armillaria ostoyae). All fungi are heterotrophic, meaning that they must acquire their food from the environment. These organisms also exhibit diversity in their specific activities, niches, and potential effects on human health. For example, some fungi are consumed or used to produce food by humans while others can cause severe or even deadly infections2.
Within kingdom Fungi, two distinct taxonomic divisions exist based on the reproductive strategy of the species. First, Ascomycota, known as the sac fungi, have a reproductive structure called an ascus. Together, many asci form the ascocarp, or &#8220;fruiting body&#8221; of the fungi. These fungi can reproduce sexually or asexually by budding. Ascomycota consist of some economically important species like the yeast used in brewing beer and baking bread, and some species popular in cuisine, such as morels and truffles. The second group, Basidiomycota, includes organisms commonly known as &#8220;club fungi&#8221; due to the presence of a large club-shaped reproductive structure called the basidium. In contrast to Ascomycota, most Basidomucota reproduce exclusively by sexual reproduction. This occurs when two haploid mycelia join to form a basidiocarp. On a button mushroom, this is the cap structure. On the underside of the basidiocarp, reproductive structures called gills lined with structures called basidia produce haploid nuclei that eventually form basidiospores. Basidiospores are then dispersed by the wind. This taxonomic division includes many commonly eaten button mushrooms, as well as wild puff mushrooms.
<h4>Importance of Microbiology</h4>
Overall, it is clear that the diverse functions and properties of microorganisms make their identification extremely important for human health and well-being. While a vast number of species of fungi and bacteria have been identified to date, it is estimated that millions more have yet to be identified and classified. In addition, human disciplines that explore Fungi and Bacteria are as diverse as the organisms themselves and will undoubtedly continue to grow. The relevance of microorganisms to the medical fields is more prevalent now then ever, as multi-antibiotic resistant species of infectious bacteria threaten human health. Understanding the structure and biological characteristics of infectious and non-infectious species provides key insights necessary for the creation of new antibiotic treatments and preventative strategies3-4.
Microbiology is also critical to the food industry. Numerous products, including yogurt, cheese, kombucha, bread, and alcohol are made using specific categories or species of microbes. At the same time, food scientists focus on engineering chemical compounds and preservatives that will reduce the growth of unwanted microbes on food. In all these processes, an understanding of how microbes survive, reproduce, and affect their environment is necessary.
<h4>References </h4>
<ol class="references">
	<li>Hiroshi Asakura, Holger Brueggemann, Sou-ichi Makino, and Yoshiko Sugita-Konishi, &#8220;Molecular Approaches for the Classification of Microbial Pathogens of Public Health Significance,&#8221; BioMed Research International, vol. 2014, Article ID 725801, 2 pages, 2014. https://doi.org/10.1155/2014/725801.</li>
	<li>Treseder, K. K. and J. T. Lennon (2015). 'Fungal traits that drive ecosystem dynamics on land.' Microbiol Mol Biol Rev 79(2): 243-262.</li>
	<li>Arvanitis, M. and E. Mylonakis (2015). 'Fungal-bacterial interactions and their relevance in health.' Cell Microbiol 17(10): 1442-1446.</li>
	<li>Aarestrup, F. M., et al. (2008). 'Resistance in bacteria of the food chain: epidemiology and control strategies.' Expert Rev Anti Infect Ther 6(5): 733-750.</li>
</ol>

Microbial Colony and Fungal Diversity - Concept

Diversità microbica e fungina

Bacteria and fungi are two highly diverse groups of organisms that can have significant beneficial or detrimental impacts on human health. For this ...

Cell Division

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Preparation of Onion Root Tips and Solutions
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">For the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water to grow roots for several days. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, clean the onion roots of any dirt or debris. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, in a 1.5 mL tube, dissolve 10 mcg of nocodazole per 1 mL of dimethyl sulfoxide solution, making 1 tube per student. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Finally, set out all of the other tools, materials, and reagents from the list of materials onto each student&#39;s lab bench. 
</div></li></ul></li></ol>

Cell Division: Observing Cell Cycle in a Root Tip - Prep

Divisione cellulare

Preparation of Onion Root Tips and Solutions
ExpandFor the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water ...

Processi cellulari

Lab Manual Sub Articles

Bacterial Transformation

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Bacterial Transformation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">First, ensure that each student or student group has a filled ice bucket.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, set up a tube rack for each work station.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Into the tube racks, place two empty, sterile 1.5 mL tubes, and then two one mL aliquots of SOC media.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, place the plasmid tube into the ice bucket and add two tubes of competent E. coli cells, allowing them to thaw if frozen.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Set out two LB agar plates per group and two LB agar ampicillin plates per group.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Ensure the students have the appropriate pipettes.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">And finally, set a water bath to 42 &#176;C and a temperature-controlled shaker to 37 &#176;C.
</div></li></ul></li></ol>

Bacterial Transformation using Plasmids - Prep

Trasformazione batterica

Bacterial Transformation
ExpandFirst, ensure that each student or student group has a filled ice bucket.
Then, set up a tube rack for each work station.
...

Cell Structure

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Visualizing Onion and Cheek Cells
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Immediately before the experiment, wash and peel onion bulbs for the class.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Remove the entire brown outer skin and cut the onion in half with a knife. Pull apart the layers of the onion. The thin, nearly transparent film layers within the onion will be used by the students.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Place the onion film into a Petri dish and then place this into the refrigerator until the students arrive.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Bring out the microscopes and set them on the lab bench.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Set the microscopes to the lowest magnification and plug them into an electrical outlet.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Clean the lenses with lens paper or laboratory wipes and test the knobs to make sure the lenses focus properly.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Make sure that the stage can be moved.
</div></li></ul></li></ol>

Cell Structure: Visualizing Onion and Human Cells - Prep

Struttura cellulare

Visualizing Onion and Cheek Cells
ExpandImmediately before the experiment, wash and peel onion bulbs for the class.
Remove the entire brown outer skin and ...