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.

以前の研究では、C1498細胞株は、急性顆粒球5、骨髄6またはNKT 7細胞白血病のインデューサーとして記載されました。しかし、文献で実証データが存在しないか、または不完全のいずれかでした。ここに提示プロトコルは、培養されたC1498細胞を特徴付けるために、それらが注入された後にマウスにおいて誘導される白血病の性質を決定するために、フローサイトメトリー、免疫蛍光、MGG染色および細胞化学的アッセイなどの様々な技術を使用します。 我々は免疫染色した後にインビトロで培養C1498細胞の表現型およびフローサイトメトリー分析時に行われたこれらの細胞は、以前に文献6,7に記載されているいくつかの細胞表面造血マーカーを発現しているため、我々はいくつかの制限を観察しました。我々の結果と一致して、ラブレらは 、フローcytometを使用C1498細胞に成熟したTCRの細胞表面発現を観察しませんでしたRY染色。それらはCD3εとTCRVβ8.2のmRNA 7を検出した後、しかし、それらは、NKT細胞株であるためにそれらを検討しました。我々はまた、細胞（&gt; 70％）のほとんどでTCRVβ鎖およびCD3ε分子の細胞内発現を観察したが、Macの-3分子の同時細胞内発現もあったので、それらの造血系統を決定することができませんでした。 ミエロペルオキシダーゼ、MGG染色および細胞化学を用いた機能性エステラーゼを分析するためのアセスメントは、C1498細胞株は骨髄起源を持っていたし、芽球と骨髄芽球から構成されていたことを実証しました。これらの結果は、フローサイトメトリー染色分析で得られたのMac-3 +細胞の割合と一致しました。定量的ではないものの、これらの手順を実行するためのキーの実験を表します。確かに、彼らは、これまでのところ、ないまたはfを表現する造血細胞の系譜と分化ステージを特徴付けるための最良の既存の方法のままEW特定の表現型マーカー。 染色フローサイトメトリーは、C1498細胞を静脈内注射した後にコンジェニックマウスにおける急性白血病の開発を実証するために役に立ちました。末梢血および様々な臓器に浸潤CD45.2 + C1498細胞を単離し、それらの周波数が決定されました。定量化はまた、免疫表現後固有髄質および脾細胞を分析するために行きました。彼らはいくつかの造血マーカーを発現したとして、C1498細胞の表現型は臓器で調べたときの制限事項が発生した（そのうちのほんの数は、B220 +でした）。観察された急性白血病の性質を定義するには、メイグリュンワルドギムザ染色し、単球及び顆粒球エステラーゼの活性の分析は、骨髄を用いて行きました。結果は、C1498細胞は骨髄単球性白血病の発症を明らかにし、彼らの骨髄芽球と単芽の形態および機能を維持したことを示しました。 <p clasS = &quot;jove_content&quot;&gt;細胞化学反応およびMGG染色を行う際のpHのエラーは結果の誤った解釈につながることができますので、このプロトコルに記載の重要なステップのための配慮では、特に注意がpHに与えられるべきです。顆粒球およびリンパ球はまた、より高いpH値で、このテストのために陽性に染色することができますので、例えば、α-ナフチル酪酸エステラーゼ活性は6.0のpHで単球細胞に特異的です。細胞を固定すると、MGG染色を行う前に、推奨、我々はC1498細胞を用いたエステラーゼ細胞化学反応を行う場合にのみCAFの固定は満足な結果を提供したことを示したされていません。フローサイトメトリーによりCD115分子とその検出の発現を維持するために、試料（ 例えば 、血液、骨髄、脾臓細胞）の全ては、手順の間、氷上に維持されるべきです。染色は、フローサイトメトリーおよび/または免疫蛍光実験を、抗体の参照、それらの記憶再において観察されていない場合表彰及びその希釈物をチェックする必要があります。材料/機器テーブルで指定された参考文献は、フローサイトメトリーまたは免疫蛍光アプリケーションに設定されています。一次/二次抗体またはその共役フルオロフォアは不適当なストレージにそれらの活性を失っている可能性がある（ 例えば 、光または熱への曝露）、不適切な希釈、広範な凍結/解凍または汚染されたバッファを使用。それらが正常に機能していることを確認するために、ポジティブコントロールを実行します。目的のタンパク質を発現することが知られているマウスの骨髄または脾臓由来の細胞を使用します。高いバックグラウンドや非特異的な染色を避けるために、細胞が適切に洗浄し、（免疫蛍光のための）高湿度に保ち、指示通り抗体が希釈されていることをされていることを確認してください。正確に試料中のバックグラウンドレベルを決定するために、アイソタイプコントロール抗体と一次抗体のための同じ濃度および希釈を使用してください。エステラーゼ細胞化学実験のために、試薬は、精製マウス脾臓顆粒球（Ly6G +）および単球（CD115 +）細胞を含む陽性および陰性対照スライドを使用して試験することができます。 この研究で記載された手順は、C1498細胞の注射後のマウスにおいて観察された白血病の特徴の多くは、ヒト急性骨髄単球性白血病11,12と共通の特徴を共有していることを示しました。侵入した白血病細胞（前駆細胞および前駆体）は、成熟および未成熟骨髄造血細胞の減少をもたらしました。単球細胞であるとしてC1498細胞は、末梢血中の高頻度（&gt; 20％）で存在します。肝腫および脾腫は、白血病細胞の浸潤から生じることが観察された、およびBリンパ球および骨髄細胞の大幅な増加も脾腫を伴うことが観察されました。血小板数は、血液分析器を用いて推定したときに血小板減少も観察されました。 それはショーでしたnは、 試験管内実験で使用して、C1498細胞は、可溶性因子13を分泌することにより、通常のマウスの造血を阻害すること。いくつかの腫瘍のマウスモデルにおいて、（単球および顆粒球細胞を含む）、未熟骨髄細胞はまた、抗腫瘍特異的T細胞活性化および増殖14を阻害する脾臓を骨髄から移行することが示されています。このように、骨髄中で観察された造血細胞の減少は、造血および/またはそれらの移民からの欠乏のいずれかから生じた可能性があります。この後者のメカニズムは、末梢血中の単球の存在または脾臓の拡大骨髄性画分の観察を説明することができます。これらの細胞は、改善された脾臓造血由来していることができることも考えられます。実際に、定常状態の条件下で、脾臓B細胞のいくつかのサブセットは、成熟Bリンパ球15の前駆体として同定されました。また、炎症性の条件の下で、のmedullary幹および前駆細胞は、成熟単球16の産生を誘導するために脾臓に移転することが示されています。このプロトコルは、私たちは、白血病の発症に関与しているメカニズムに関する結論を引き出すことはできません。また、付加的な機能だけでなく、分子アッセイは、そうするために採用されるべきです。しかし、これらのデータは、急性骨髄性白血病の臨床的特徴についての詳細な情報が含まれており、研究者が新たな治療薬の効果を評価し、理解するのに役立ちます。

急性骨髄性白血病（AML）は、成熟の異なる段階でブロックされた造血骨髄細胞の制御されない増殖を特徴とします。この調節不全は、顆粒球、単球、赤血球またはmegaryocytic分化経路1に影響を与えることができます。 AML細胞は、血小板減少、リンパ球減少症および貧血になる造血障害につながる、骨髄に蓄積します。白血病細胞は、血液および非リンパ系器官に侵入します。 C1498マウスモデルは、文献には、血液中に侵入を記述するがん細胞が1941年に白血病10ヶ月齢のC57BL / 6（H-2 b）の雌性マウスから単離されたので、急性白血病のためのモデルとして何十年も使用されてきました増殖性の高いC1498細胞による、造血器官（ 例えば 、脾臓およびリンパ節）および非造血器官（ 例えば 、肝臓、肺、卵巣、及び腎臓）それらが中を経由して注入した後に感受性マウス2-4にtravenous、皮下または腹腔内経路。しかし、このマウスモデルは、2,5顆粒球または骨髄のいずれか6白血病を誘発することが報告されました。より最近では、2002年に発表された研究は、マウスNKT細胞白血病7などの癌のこのタイプを記載しました。このように、文献はこのC1498細胞株の性質と、それは、マウスにおいて誘導関連白血病に関する異なります。 70年代 - これらの矛盾は、詳細の欠如および細胞に関する更新公開されている情報と多くの研究は、1950年に行われたため、一般的には白血病疾患が主な原因です。 ここでは、C1498細胞を特徴付けるし、マウスにそれらの静脈内注射によって誘発される白血病疾患の性質を分析する方法を説明するための詳細なプロトコルを提供します。このプロトコルの最初のセクションは、 インビトロで培養されたC1498細胞の説明に捧げられています。蛍光抗表面および細胞内の造血マーカーに対して指向体は、フローサイトメトリーを用いてそれらの表現型を決定するために使用しました。ミエロペルオキシダーゼの存在は、免疫蛍光顕微鏡を用いて評価し、その造血系及び分化段階はエステラーゼの活性を評価するために細胞化学を用いて評価し、メイグリュンワルドギムザ染色を行いました。 C1498細胞は、その後マウスに注射し、そして誘導された急性白血病の疾患は、この原稿の第二の部分に記載されています。同様の技術は、周波数および骨髄中の白血病および固有細胞、末梢血、脾臓および非造血器官（肝臓、肺）の表現型を決定するために使用しました。 このプロトコルは、非常に再現性があり、ここで提示されたデータは、研究者が新たな治療戦略の効果を評価するのに役立ちます。この白血病のマウスモデルは、すでに免疫療法に近づく試験するために使用されていますND異なる癌化学療法薬8,9。それらの有効性は、腫瘍負荷と生存率の変化を決定することによって評価しました。このプロトコルは、治療中の白血病および他の造血細胞集団の分布と生存についての追加情報を提供するために使用することができます。

<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

1941これらの注射は、急性白血病の発症をもたらすので、同系またはジェニックマウスへのC1498細胞の静脈内注射が行われています。しかし、この病気の性質は、文献に十分に文書化されていません。ここでは、 インビトロでの C1498細胞を特徴付けるためのin vivoで誘導される白血病の性質を決定するための技術的なプロトコルを提供します。この手順の最初の部分は、造血系および培養C1498細胞の分化段階を決定することに焦点を当てています。これを達成するために、マルチパラメトリックフローサイトメトリー染色は、造血細胞マーカーを検出するために使用されます。免疫蛍光顕微鏡法、細胞化学およびメイグリュンワルドギムザ染色は、その後、それぞれ、ミエロペルオキシダーゼの発現、エステラーゼおよび細胞形態の活性を評価するために行われます。このプロトコルの第2の部分は、 内に誘導される白血病疾患を記述するに専用されています生体内。後者は、血液中の白血病および内在細胞の頻度を決定することによって達成造血器官（ 例えば 、骨髄および脾臓）および非リンパ組織（ 例えば 、肝臓および肺）特異的染色を使用してフローサイトメトリー分析を流すことができます。白血病の性質は、その後、骨髄中の特定のエステラーゼのためのメイ・グリュンワルドギムザ染色および染色を用いて確認されます。ここでは、年齢をマッチさせたC1498-とPBSを注射したマウスでは、このプロトコルを用いて得られた結果を示します。

C1498マウスモデルを特徴づけるために、我々は、2つの主要な手順で進めました。まず、C1498細胞は、 インビトロでの造血系および成熟段階（ 図1）を決定するために特徴づけました。これらの細胞は、その後、コンジェニックマウスに注射し、および誘導白血病疾患の性質は、異なる特徴を決定するために評価した。白血病細胞浸潤、それらの表現型は、骨髄中の造血細胞（成熟及び前駆細胞/前駆体）の定量化、周波数C1498細胞や血液や臓器の腫脹（脾臓、肝臓、および肺）と細胞組成の評価で成熟造血細胞の。 インビトロでの C1498細胞の表現型を特徴づけるために、細胞は、造血前駆体および成熟細胞（ 表1）で表される分子に対する抗体で標識し、その結果を流れcytometを用いて分析しました。RY。 C1498細胞は、Mac-1（CD11bの/ CD18）（約7％）、B220（&gt; 25％）の細胞表面発現について陽性であり、それらはCD3ε、T細胞受容体（TCR）のVβ鎖およびMacの細胞内発現を示し-3（ 図2AおよびB）。細胞は、細胞表面マーカーLy6G、Ly6C、CD115、CD21 / CD35、CD19、CD3、CD4、CD8、NK1.1、および汎NK分子についてとCD4とCD8の細胞内発現について陰性であった（データは示していません） 。次いで、それらを、造血幹細胞および前駆細胞（ 表1）のマーカーを調べました。彼らはまた、CD117、CD34、SCA-1、CD150およびCD16 / 32の細胞表面発現について陰性であった（データは示さず）。これらの白血病細胞は、その後、接着、抗原の提示および共刺激分子の発現を決定するために試験しました。細胞は、表面マーカーLFA-1（のCD11a / CD18）、CD44、CD31（PECAM-1）、及びH-2D bは発現され、CD80、CD86およびCD274（データは示していない）、MHCクラスII陰性でした。 C1498細胞トンherefore骨髄（マック-1、Macの-3）およびリンパ系マーカー（B220、CD3、TCR）の両方を発現していました。 より自分の造血系統を特徴づけるために、ミエロペルオキシダーゼの発現は、免疫蛍光顕微鏡を用いて評価しました。細胞のすべてが彼らの骨髄起源（ 図3A）を検証ミエロペルオキシダーゼ、陽性でした。細胞の大部分はまた、αナフチル酪酸エステラーゼ（ 図3B、左パネル ）について陽性染色され、そのうちのいくつかは、ナフトールAS-Dクロロ酢酸エステラーゼ（黒矢印）（ 図3B、右パネル ）について染色しました。結果は、細胞が単球および顆粒細胞の混合物を含有していることを示しています。メイグリュンワルドギムザ染色を行った後、C1498細胞は、核内で3〜5核小体、核周囲ハロ、多数の空胞及び好塩基性細胞質（ 図3C、高い核-細胞質比でブラスト様形態を表示することが観察されました）。目私たちは、C1498細胞株は、単芽球と骨髄芽球で構成されています。 C1498細胞（CD45.2 +）を、静脈内でCD45.1 +マウスに注射しました。マウスは、細胞を注入した17〜19日後に死亡しました。彼らは病気で死亡前に白血病のタイプを分析することができるように、これらのマウスを屠殺しました。 PBSを注射した対照マウスは、比較のために同一の時点で分析しました。 C1498細胞を注射したマウスは、メイグリュンワルドギムザ染色が（ 図4A）を実施した後の細胞の爆発のような外観によって示されるように、彼らの骨髄にC1498細胞の大量の浸潤を示しました。彼らはまた、急性骨髄性白血病の特徴である単芽球と骨髄芽細胞の蓄積を実証し、その単球及び顆粒球の表現型（ 図4BおよびC）を保存しました。 Dに骨髄造血細胞の数は、白血病細胞の浸潤、CD45.2 + C1498細胞、Bリンパ球、単球及び（前駆細胞、前駆体および成熟した細胞を含む）、顆粒球集団以下低かった免疫蛍光染色およびサイトメトリー分析マルチパラメトリックフローを用いて定量したかどうかをetermine。白血病細胞は、造血細胞（データは示していない）の16〜36％を占めました。他の細胞型は、全ての平均で顆粒球細胞および3倍の平均の4倍、B細胞サブセットの平均で5倍（PBSを注射したマウスよりもC1498を注射したマウスにおいて有意に低い数字に存在しましたCへの単球サブセットのために）（ 図5A）。 白血病と対照マウスの血液試料中の単核細胞の頻度の調査は、それらがリンパ球（ 図6A）の匹敵する割合が、単球の高い周波数を含むことを示しましたそして白血病細胞。これらの特性は、急性骨髄性白血病11（ 図6B）の代表です。 急性骨髄単球性白血病12の他の機能の中に、C1498-注射したマウスは、腫れ肝臓（肝腫）、肺および脾臓（脾腫）（ 図7A）を提示します。 CD45.2 + C1498細胞の様々な周波数は、免疫蛍光染色を使用して、これらの器官において検出され、解析（ 図7B）フローサイトメトリーました。脾腫浸潤した単球の高い数に起因することができ、我々はまた、脾臓集団の割合を推定しました。 Bリンパ球、単球内の細胞および顆粒細胞画分の数は2倍の平均値で、有意に大きかった、2.5倍と3倍、それぞれ、制御脾臓（ 図7C）に比べて白血病脾臓インチ薄ページ= &quot;1&quot;&gt; <img alt="図1" src="/files/ftp_upload/54270/54270fig1.jpg" /> 図1. 体外培養C1498細胞株および急性白血病のin vivoでの説明に特徴付けるためのプロトコルのセットアップの概略図。造血系および組織培養C1498細胞の分化段階を最初に決定しました。 C1498細胞は、その後、急性白血病の発達を誘導するためにコンジェニックマウスに注射しました。骨髄、末梢血、脾臓、肝臓および肺組織の単離は、C1498細胞浸潤後の周波数、表現型および形態学的変化を決定しました。 IV：静脈内MGG：メイグリュンワルドギムザ<a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <html>広告/ 54270 / 54270fig2.jpg &quot;/&gt; in vitroで培養。ドットプロットおよび細胞表面（A）および細胞内（B）のヒストグラム代表的なフローサイトメトリー後のC1498細胞の図2.表現型分析は、造血成熟細胞分化に関連した分子が示されているC1498-表明しました。 C1498細胞は、細胞表面のCD11b、CD18およびB220マーカーまたはそのアイソタイプコントロールに特異的であった蛍光抗体を用いて、培養物から回収し、洗浄し、標識しました。細胞内染色のために、細胞を固定し、透過およびMac-3に対する抗体、CD3ε、およびTCR（T細胞受容体）のVβ鎖またはそのアイソタイプコントロールの共通のエピトープを用いて標識しました。分析は、生きた細胞でゲーティングを使用して実施した。 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <html> ntent「FO：キープtogether.withinページ= &quot;1&quot;&gt; <img alt="図3" src="/files/ftp_upload/54270/54270fig3.jpg" /> 図3.機能および培養C1498細胞の形態学的特徴付け 。 C1498細胞を培養物から採取し、顕微鏡用スライド上に遠心分離した。（A）ミエロペルオキシダーゼの発現のために染色が免疫蛍光を用いて行った。（B）細胞化学反応は、αナフチル酪酸エステラーゼ（NBE）およびナフトールAS-Dクロロアセテートエステラーゼを分析するために使用しました。 （CAE）C1498細胞における活性。細胞は茶色と赤紫色、大型の細胞質顆粒は、それぞれ、観察された各ラベルについて陽性であると考えられた。C1498細胞の（C）、メイグリュンワルドギムザ（MGG）染色。各染色実験では、顕微鏡対物倍率が示されています。各画像は、3つの別々の実験の代表です。large.jpg &quot;ターゲット=&quot; _空白 &quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図4" src="/files/ftp_upload/54270/54270fig4.jpg" /> 図4.骨髄、PBS中のモルフォロジーおよびC1498-注射したマウス。骨髄細胞を、PBSおよびC1498細胞を注射したマウスから単離し、顕微鏡用スライド上に遠心分離した。（A）メイグリュンワルドギムザ（MGG）染色。（B ）α-ナフチル酪酸エステラーゼ（NBE）および（C）ナフトールAS-Dクロロエステラーゼ（CAE）関数は、細胞化学を用いて評価しました。パネルAでは、バンド（未成熟）またはセグメント（成熟した）好中球は、PBSを注射したマウスよりもC1498-注射したマウスの骨髄中の見えにくいです。パネルBおよびCは、制御骨髄において観察された数字と比較すると、白血病、骨髄中の単球や顆粒球細胞の蓄積があったことを示しています。すべて顕微鏡分析は、100X倍率の対物レンズを用いて行った。 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図5" src="/files/ftp_upload/54270/54270fig5.jpg" /> 髄様、PBS中の集団とC1498-注射したマウスの図5.定量分析。骨髄細胞を、PBSおよびC1498細胞を注射したマウスから単離し、細胞計数を行った後に推定しました。異なる細胞集団の頻度は、免疫染色及びサイトメトリー分析生細胞ゲーティング流れ後に測定した。（A）B細胞サブセットは、中のBリンパ球（B）、顆粒球細胞を成熟プロB細胞の段階でCD19 + B220 +細胞を含まCD3 -前駆体Aを含めてのCD11b + Ly6G +系統、ND未熟および成熟顆粒（C）単球サブセットはCD3のように定義された- 。CD115 +および単球ステージを成熟させる前駆内のセルが含まれていました。 n = 7匹のマウス/群、およびデータは、平均±SEMを示すヒストグラムとして提示されています。 ***はp &lt;0.0001 **、P &lt;0.01、PBS処置およびC1498-注射したマウスを比較した対応のないスチューデントt検定。 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図6" src="/files/ftp_upload/54270/54270fig6.jpg" /> PBS処置およびC1498-注射したマウスにおける単核細胞サブセットの図6.血液分析 。それぞれ、PBSおよびC1498の中でCD3 +およびB220 +細胞と定義した（A）TおよびBリンパ球の割合、のドットプロット代表的なフローサイトメトリー細胞を注射しました。及びCD115 + Ly6C high細胞-マウス（B）C1498の白血病とコントロールにおける単球細胞の頻度は、（PBS）マウスは、CD115 + Ly6Cを分析することによって決定しました。分析は、生細胞をゲーティングすることによって行いました。白血病マウスおよび対照マウスを比較するために、CD45.2 + C1498細胞が除外された。 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig6large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図7" src="/files/ftp_upload/54270/54270fig7.jpg" /> 白血病およびコントロールマウスにおける脾臓集団の図7.推定。 （A）は、肝臓、肺および脾臓の代表的な写真は、対照マウスと比較して、白血病マウスの腫脹。脾臓を採取し、秤量し、脾細胞を組織破壊、次のカウントしたした。デフにおける白血病細胞の頻度を表す（B）ヒストグラムを免疫染色は、CD45.2 +細胞について実施し、結果は、フローサイトメトリーを用いて分析した。（C）脾臓Bの試算、顆粒球及び単球細胞数免疫染色した後、分析ゲートフローサイトメトリー後erent器官、生CD19 + B220 +を同定しました、CD3 -のCD11b + Ly6G +、CD3 -のCD11b + Ly6C -およびCD3 -のCD11b + Ly6C high細胞。肺、脾臓および肝臓のために示されたスケールバーは1cmに示しています。 n = 5から8マウス/群、およびデータは、平均±SEMとしてヒストグラムで表現されている*はp &lt;0.05; PBS処置およびC1498-注射したマウスを比較**は p = 0.0033、対応のないスチューデントのt検定。。 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig7large.jpg" target="_blank">してくださいこの図の拡大版をご覧になるにはこちらをクリックしてください。</a> <table fo:keep-together.within-page="1"><tbodY&gt; <tr><td> 細胞型 </td><td> 膜または細胞内分子 </td></tr><tr><td></td><td></td></tr><tr><td> 前駆体および成熟した細胞 </td><td></td></tr><tr><td> NK細胞</td><td> NK1.1 +、パンのNK + </td></tr><tr><td> NKT細胞</td><td> NK1.1 +、汎NK +、TCR Vbeta +（8.2）、CD3 + </td></tr><tr><td> Tリンパ球</td><td> TCR Vbeta +、CD3 +、CD4 +、CD8 + </td></tr><tr><td> B細胞前駆体およびBリンパ球</td><td> B220 +、CD19 +、CD21 / 35 + </td></tr><tr><td>顆粒球前駆体および顆粒球</td><td> Ly6G +、Macの-1 +、CD11bの+ </td></tr><tr><td>単球前駆体及び単球/マクロファージ</td><td> CD11b +、Macの-1 +、Macの-3 +、CD21 / 35 +、CD115 +、Ly6Chi </td></tr><tr><td></td><td></td></tr><tr><td> 前駆細胞 </td><td></td></tr><tr><td>多能性前駆細胞</td><td> CD117 +のSca-1+ CD34 +（LIN-CD150-） </td></tr><tr><td>リンパプライミング多能前駆細胞</td><td> CD117hi SCA-1hi CD127 +（LIN-） </td></tr><tr><td>リンパ球系共通前駆細胞</td><td> CD117lo SCA-1LO CD127 +（LIN-） </td></tr><tr><td>骨髄系共通前駆細胞</td><td> CD16 / 32lo CD117 + CD34int（LIN-SCA-1-） </td></tr><tr><td>顆粒球 - マクロファージ前駆細胞</td><td> CD16 / 32hi CD117 + CD34hi（LIN-SCA-1-） </td></tr><tr><td>巨核球、赤血球前駆細胞</td><td> CD16 / 32lo CD117 + CD34lo（LIN-SCA-1-） </td></tr><tr><td></td><td></td></tr><tr><td> 造血幹細胞 </td><td> CD117 + SCA-1 + CD150 +（LIN-CD34-） </td></tr></tbody></table> 表造血細胞系統および分化の1マーカー。 CD：CD分類。林：成熟細胞のマーカー; LO：低発現;こんにちは：高発現; int型：中間式。 NK：ナチュラルキラー細胞; TCR：T細胞受容体。

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

この原稿は、in vitroおよびその注射後にマウスにおいて誘導される急性白血病に C1498細胞培養物を特徴付けるために使用することができる技術的手順を提供します。表現型および機能的分析は、フローサイトメトリー、免疫蛍光顕微鏡法、細胞化学およびメイグリュンワルドギムザ染色を使用して実行されます。

マウスC1498細胞株およびその関連白血病マウスモデルを特徴付けるための詳細なプロトコル

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

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Cancer Research

19.9K ビュー.  INSERM, UMR-S-1172, Centre de Recherche Jean-Pierre Aubert (JPARC). この原稿は、in vitroおよびその注射後にマウスにおいて誘導される急性白血病に C1498細胞培養物を特徴付けるために使用することができる技術的手順を提供します。表現型および機能的分析は、フローサイトメトリー、免疫蛍光顕微鏡法、細胞化学およびメイグリュンワルドギムザ染色を使用して実行されます。 

ビデオ: 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

Balmerシリーズ

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 ...

Education

JoVE Lab Manual

化学

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

生物の遺伝学

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

生物学

遺伝学

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

動物の行動

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 ...

生態学

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

ハーディ・ワインバーグと遺伝的ドリフト

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 ...

進化論

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

進化的関係

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

動物の多様性

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

微生物と真菌の多様性

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

細胞分裂

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 ...

細胞プロセス

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

細菌の形質転換

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

細胞構造

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