אנו מודים טראסט לחינוך סביבתי שירותים האגודה, ביולוגיה סינתטית בקרן קיימברידג SynBio ומשרד צדק חברתי והעצמה, ממשלת הודו, עבור תמיכה כספית.

<ol>
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The authors declare that they have no competing financial interests.

השלבים הקריטיים ביותר בדור של מוטציות לא מסומנות הם: 1) עיצוב פלסמיד זהיר כדי להבטיח את אזור המיקוד רק משתנה; 2) להבטיח כי דגימות להישאר axenic, במיוחד כאשר בתרבית על סוכרוז; 3) ציפוי טרנספורמציה תאים עבור דור מוטציה מסומן בתחילה על צלחות אגרו BG11 חסרי אנטיביוטיקה, ואחריו תו?...

כחוליות מהוות מערכה עתיקה ומגוונת אבולוציונית של חיידקים נמצאים כמעט בכל סביבה טבעית על פני כדור הארץ. בשנת מערכות אקולוגיות ימיות הם במיוחד שופע לשחק תפקיד מפתח במחזורים מזין רבים, והיוו כמחצית קיבוע פחמן 1, רוב קיבוע חנקן 2 ומאות מיליוני טונות של ייצור פחמימנים 3 באוקיינוסים בשנה. כלורופלסטים, אברון האחראי על הפוטוסינתזה אצות איקריוטיים וצמחים, צפויים התפתחו מתוך cyanobacterium כי נבלע על ידי הפונדקאי 4. כחוליות הוכיחו אורגניזמים מודל שימושי לחקר הפוטוסינתזה, אלקטרון התחבורה 5 ו הביוכימיים, שרבים מהם שמורים בצמחים. בשנת כחוליות בנוסף יותר ויותר בשימוש לייצור מזון, דלק ביולוגי 6, חשמל 7 ותעשייתיים תרכובות 8, בשל היי שלהםהמרה יעילה ghly מים ו- CO 2 עד ביומסה באמצעות אנרגיה סולארית 9. מינים רבים יכולים להיות מעובד על קרקע שאינה חקלאית עם חומרים מזינים מינימאליים ומי ים, דבר המצביע על כך כחוליות עלולים להיות מבוגרים בקנה מידה גדולה מבלי להשפיע ייצור חקלאי. מינים מסוימים הם גם מקורות של מוצרים טבעיים, כולל תרכובות נגד פטריות, אנטיבקטריאלי ואנטי סרטן 10,11. היכולת ליצור מוטציות היא מפתח להבנת פוטוסינתזה כחוליות, ביוכימיה ופיזיולוגיה, וחיוני להתפתחות של זנים למטרות תעשייתיות. רוב המחקרים שפורסמו גנרטור מהונדס גנטי זנים ידי החדרת קלטת עמידות לאנטיביוטיקה לתוך האתר של עניין. זה מגביל את מספר מוטציות יכולות להיות מוחדר זן, כמו שרק כמה קלטות עמידות לאנטיביוטיקה זמינות לשימוש כחוליות. זנים המכילים גנים המקנות מחדש אנטיביוטיsistance לא יכול לשמש לייצור תעשייתי בבריכות פתוחות, אשר צפוי להיות אמצעי היחיד החסכוני לייצר דלק ביולוגי ומוצרים בעלי ערך נמוך אחרים 12. הדור של מוטציות לא מסומנות מתגבר על מגבלות אלה. מוטציות לא מסומנות אינם מכילים דנ&quot;א זר, אלא אם כן נכלל בכוונה, וניתן לטפל מספר פעמים. לכן אפשר ליצור שינויים רבים ב זן כפי רצויות. בנוסף, תופעות קוטב על גנים במורד הזרם של אתר שינוי ניתן למזער, המאפשר שינוי מדויק יותר של האורגניזם 13. כדי ליצור זנים מוטנטים, פלסמידים התאבדות המכיל שני קטעי דנ&quot;א זהה אזורים בכרומוזום כחוליות איגוף הגן למחיקה (שמכונה 5 &#39;ו 3&#39; אזורי איגוף) בנויים ראשון. שני גנים ואז מוכנס בין אזורי איגוף אלה. אחד מהם מקודד חלבון עמידות לאנטיביוטיקה; השני מקודד SacB, אשר לדרבןuces levansucrase, תרכובת מקנה רגישה סוכרוז. בשלב הראשון של התהליך, מוטציות מסומנות, זנים כלומר המכילים כמה דנ&quot;א זר, נוצרות. הבונה פלסמיד הוא מעורבב עם התאים כחוליות והדנ&quot;א נלקח באופן טבעי על ידי האורגניזם. Transformants נבחר על ידי צמיחה על צלחות אגרו המכילות את אנטיביוטי המתאים ואת הגנוטיפ המוטציה מאומת על ידי PCR. פלסמידים התאבדות לא יכולים לשכפל בתוך הזן של עניין. לכן כל מושבות עמידות לאנטיביוטיקה תגרומנה מאירוע רקומבינציה לפיה הגן של עניין מוכנס לתוך הכרומוזום. כדי ליצור מוטציות לא מסומנות, המוטציה הניכרת הוא מעורב אז עם פלסמיד התאבדות נוסף שהכיל רק 5 &#39;ו 3&#39; אזורי האיגוף. עם זאת, אם החדרת דנ&quot;א זר נדרשה, פלסמיד המורכב של 5 &#39;ו 3&#39; איגוף אזורים עם קסטה המכילה את הגנים של העניין המוכנס בין קטעי דנ&quot;א אלה, ניתן להשתמש. Sele גודלction היא באמצעות צמיחה על צלחות אגר המכיל סוכרוז. כמו סוכרוז הוא קטלני לתאים כאשר מוצר גן sacB מתבטא, התאים היחידים ששורדים הם אלה שבם אירוע רקומבינציה שני התרחש, לפיה גן רגישות סוכרוז, בנוסף גן העמידות לאנטיביוטיקה, כבר recombined מתוך כרומוזום על פלסמיד. כתוצאה מכך של חילופי recombinational, אזורי האיגוף וכל DNA ביניהם מוכנסים לתוך כרומוזום. השתמשנו בהצלחה בשיטות אלה כדי ליצור מוטציות כרומוזומליות מרובות באותו הזן של Synechocystis sp. PCC6803 (להלן המכונה Synechocystis) 13,14, להציג מוטציות נקודה אחת לתוך גן של עניין 13 ו לביטוי של קלטות גן. בעוד הדור של knockouts המסומנת הודגם לפני עבודתנו Synechocystis 15,16, שיטה מפורטת, שנעזרמצגת ויזואלית של השלבים הקריטיים, הוא לא זמין לציבור. גם יש לנו ליישם אותה השיטה לייצור של knockouts ניכרה cyanobacterium מודל אחר, sp Synechococcus. PCC7002 (להלן המכונה Synechococcus). פרוטוקול זה מספק שיטה ברורה, פשוטה ליצירת מוטציות ובפרוטוקול מהיר על תיקוף ואחסון זנים אלה.

<table border="0" cellpadding="0" cellspacing="0" width="645">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:237px;">NaNO3</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">S5506</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4.7H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">230391</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">C1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">citric acid</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">C0759</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2EDTA</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">EDT002</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">H3BO3</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">339067</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MnCl2.4H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">M3634</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ZnSO4.7H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">Z4750</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2MoO4.2H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">331058</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CuSO4.5H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">209198</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Co(NO3)2.6H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">239267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ferric ammonium citrate</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">F5879</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">K2HPO4</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">P3786</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2CO3</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">SODC001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TES</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">T1375</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">SODH001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cyanocobalamin</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">47869</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2S2O3</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">72049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Bacto agar</td>
			<td style="width:123px;">BD</td>
			<td style="width:119px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Sucrose</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">SUC001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Petri dish 90 mm triple vented</td>
			<td style="width:123px;">Greiner</td>
			<td style="width:119px;">633185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 &#181;m filters</td>
			<td style="width:123px;">Sartorius</td>
			<td style="width:119px;">16534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">100 mL conical flasks</td>
			<td style="width:123px;">Pyrex</td>
			<td style="width:119px;">CON004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Parafilm M 100 mm x38 m</td>
			<td style="width:123px;">Bemis</td>
			<td style="width:119px;">FIL003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phusion high fidelity DNA polymerase&#160;</td>
			<td style="width:123px;">Phusion</td>
			<td style="width:119px;">F-530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td style="width:123px;">Melford</td>
			<td style="width:119px;">MB1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA purification kit&#160;</td>
			<td style="width:123px;">MoBio</td>
			<td style="width:119px;">12100-300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Restriction endonucleases</td>
			<td style="width:123px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">T4 ligase</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:119px;">EL0011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Luria Bertani broth</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">12795-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">MES</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">M8250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Kanamycin sulfate</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">60615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ampicillin</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">GeneJET plasmid miniprep kit</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:119px;">K0503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">14 mL round-bottom tube</td>
			<td style="width:123px;">BD falcon</td>
			<td style="width:119px;">352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">GoTaq G2 Flexi DNA polymerase</td>
			<td style="width:123px;">Promega</td>
			<td style="width:119px;">M7805</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">425-600 &#181;m glass beads</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">G8772</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glycerol</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">DMSO</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Fluorescent bulbs</td>
			<td style="width:123px;">Gro-Lux</td>
			<td style="width:119px;">69</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">HT multitron photobioreactor</td>
			<td style="width:123px;">Infors</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of marked and markerless mutants in model cyanobacterial species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

עיצוב פלסמיד הוא קריטי עבור דור מוצלח של שני מוטנטים מסומנים ולא מסומנים. איור 1 נותן דוגמא של פלסמיד ו- B השתמשו כדי ליצור מוטציה מחיקה בגני Synechocystis cpcC1 ו cpcC2 13. בכל מקרה 5 &#39;ו 3&#39; אזורי האיגוף כ 900-1,000 נ&quot;ב. אזורי איגוף מופ?...

Watch this Scientific Journal Video about Generation of Marked and Markerless Mutants in Model Cyanobacterial Species at JoVE.com

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

הדור של מסומנים מוטאנטים Markerless במודל כחוליות המינים

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

generation-marked-markerless-mutants-model-cyanobacterial

Research

JoVE Journal

Biology

11.9K Views.  University of Cambridge. Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

Video: Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

פרדיגמות אפיון תרופתי של C. elegans מוטאנטים הילוכים Synaptic

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit מבחני עבור התקפים של אפילפסיה של מוטאנטים תסיסנית</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

השתלת עדשה של דג הזברה Application שלה ניתוח של מוטאנטים העין

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

שיער פולשנית דגימה טכניקה לקבל DNA באיכות גבוהה מ יונקים קטנים החמקמק

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

דור, טיהור, ואפיון של מיני חלבון DISC1 הסלולריים פולשניים

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

איתור, חזותי וquantitating הדור של מינים חמצן תגובתי במערכת מודל אמבה

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

מהירה ויעילה דג הזברה Genotyping באמצעות PCR עם רזולוציה גבוהה ממיסים ניתוח

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

תאי גזע יעיל דור מושרה אדם פלוריפוטנטיים מתאים הסומטיים של אדם עם סנדאי וירוס

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

במשובט מעקב vivo של גזע hematopoietic ובתאים המסומנים על ידי חמישה חלבונים ניאון באמצעות Confocal וmultiphoton מיקרוסקופית

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

אנליזה microbiota באמצעות שני שלבים PCR ו-הדור הבא של 16 rRNA גן רצף

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...