リサ・クレツジの批判的なフィードバックと原稿の編集に感謝します。私たちは、行動脳の灌流と細胞カウントを支援する上で非常に貴重だったクルス・マルティン研究所のすべての研究アシスタントに感謝します。三極電極の設計に関するアンジェイ・チェッチ、コンフォーカル顕微鏡の使用に対するトッド・ブルーテとボストン大学生物学イメージングコアに感謝します。この研究は、NARSADヤング・サーポジスト・グラント(AC-M、#27202)、ブレントン・R・ルッツ賞(ALC)、I.アルデン・マッキ賞(ALC)、NSF NRT UtB:神経フォトニクス国立研究フェローシップ(ALC、#DGE1633516)、ボストン大学学部研究機会プログラム(WWY)によって支援されました。資金提供者は、研究デザイン、データ収集と分析、出版の決定、原稿の作成に何の役割も持っていませんでした。

<ol>
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著者らは競合する利益を宣言しない。

本明細書において、前頭皮質ニューロンの大きな集団における関心のある新規遺伝子の操作をマウスにおける行動アッセイと組み合わせたパイプラインが記載されている。さらに、このパイプラインは、出生後の早期発達と成人期の両方で同じマウスの行動の縦断的研究を可能にする。この技術は、時間と費用の面でコストがかかる可能性のある遺伝的動物モデルに依存する必要性をバイパ?...

ゲノムワイド関連研究(GWAS)は、脳病理1、2、3、4に関連する新しい候補遺伝子の発見を推進してきた。これらの研究は、統合失調症(SCZ)などの壊滅的な神経精神疾患を理解する上で特に有益であり、新しい遺伝子の調査は、新しい研究ラインと治療介入の開始点として役立っています5,6.SCZに対するリスクを抱える遺伝子は、出生前および出生後早期の発達の間に前頭前野(PFC)において偏った発現を示し、いくつかの神経精神疾患の病理に関与する領域7。さらに、精神障害のマウスモデルはPFCネットワーク6、8、9において異常な活動を示す。これらの結果は、SZC関連遺伝子がこの領域の発達配線に役割を果たす可能性があることを示唆している。動物モデルを用いたさらなる調査は、PFCにおける接続の確立に対するこれらの候補遺伝子の寄与を理解し、これらの遺伝子が神経精神疾患の病因に原因となる役割を有するかどうかを判断するために必要である。出生前および出生後の発達の間に特定の神経回路の遺伝子発現変化を研究することを可能にするマウスの遺伝子操作技術は、遺伝子発現変化をPFC機能不全に結びつける分子メカニズムを理解するための有望な方法である。
遺伝的マウスラインは、脳の発達と機能に対する特定の遺伝子の影響を研究する方法を提供します。しかし、トランスジェニックマウスに頼ることは、特定の遺伝子が神経回路の発達に及ぼす影響を調べるために必ずしも市販のラインが存在するとは限らないので、制限することができます。さらに、カスタムマウスラインを開発するには非常にコストがかかり、時間がかかる場合があります。トランスジェニックマウスとウイルスアプローチを組み合わせた交差遺伝子操作戦略の使用は、脳10、11、12の理解に革命を起こしている。多くの進歩にもかかわらず、ウイルス戦略は、ウイルス発現13およびウイルス発現14に関連する細胞毒性を制限することができる包装能力の限界を含むウイルスベクタータイプに依存する一定の制限を伴う。さらに、ほとんどの実験条件では、アデノ関連ウイルス(AAV)を用いた堅牢な遺伝子発現には約2~4週間の15週間が必要であり、出生後の早期開発中に遺伝子を操作することは不可能な日常的なウイルス戦略を作る。
子宮エレクトロポレーション(IUE)では、蛍光標識および薬理遺伝学的または光遺伝学的アプローチと相まって、神経回路の機能を解剖するための強力なプラットフォームを提供する、迅速かつ安価な遺伝子導入16、17を可能にする代替アプローチである。さらに、CRISPR-Cas9ゲノム編集遺伝子の開発により、細胞型特異的なノックダウンまたは特定遺伝子のノックアウトまたはプロモーター18、19の調節を介して過剰発現または正確に改変することができる。IUEを用いた遺伝子操作アプローチは、狭い発達窓20の間に遺伝子が神経回路に及ぼす影響をテストする必要がある場合に特に有利である。IUEは多目的な技術であり、特定のプロモーターの下で発現ベクターに遺伝子を挿入することによって容易に過剰発現を達成することができる。遺伝子発現のさらなる制御は、異なる強みのプロモーターを用いて発現を駆動するか、または遺伝子発現21,22を時間的に制御できる誘導性プロモーターを用いて達成できる。さらに、IUEは、特定の皮質層、細胞タイプおよび脳領域内の細胞の標的化を可能にするが、これは他のアプローチ5,17を使用して必ずしも実現可能ではない。より効率的な電界分布を発生させる3つの電極の使用に基づくIUE構成の最近の進歩は、この方法の機能的レパートリーを拡大し、科学者が新しい細胞タイプを標的にし、23、24を標的とすることができる細胞の効率、精度、および数を増加させることができた。この技術は、補体成分4A(C4A)、SCZに連結された遺伝子、PFC機能および早期認知5における原因の役割を決定するために最近使用された。
ここでは、PFCを含む前頭皮質の興奮性ニューロンの大規模な集団を標的とする遺伝子導入アプローチと、細胞および回路レベルの変化の研究を可能にするだけでなく、早期産後の発達および成人期を通じて行動を監視することを可能にする行動パラダイムを組み合わせた実験的パイプラインです。第1に説明する、前頭皮質領域における層(L)2/3錐体ニューロンの大きな集団を両側にトランスフェクトする方法である。次に、若年マウスおよび成体マウスにおける社会的行動をアッセイする課題について概説する。細胞数は、細胞トランスフェクションの範囲と位置を定量化する行動タスクの完了時に得ることができます。さらに、トランスフェクトされた細胞の数は、より多くのトランスフェクトされた細胞が行動のより大きな摂動につながるかどうかを判断するために行動データと相関させることができる。

<table border="0" cellpadding="0" cellspacing="0" style="width:664px;" width="663">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">13mm Silk Black Braided Suture</td>
			<td style="width:121px;">Havel&#39;s</td>
			<td style="width:161px;">SB77D</td>
			<td style="width:167px;">Suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adson Forceps</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">11006-12</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">C270 Webcam</td>
			<td style="width:121px;">Logitech</td>
			<td style="width:161px;">N/A</td>
			<td>Record behavior</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Electroporator</td>
			<td style="width:121px;">Custom-built</td>
			<td style="width:161px;">N/A</td>
			<td>See Figure 1 and 2 and Bullmann et al, 2015</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-500 Spin Column Plasmid DNA Maxi-preps Kit, 20preps</td>
			<td>Bio Basic Inc.</td>
			<td style="width:161px;">BS466</td>
			<td>Pladmid preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fast Green FCF</td>
			<td style="width:121px;">Sigma</td>
			<td>F7252-5G</td>
			<td>Dye for DNA solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fine scissors- sharp</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">14060-09</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand Gauze Sponges</td>
			<td>Fisher Scientific</td>
			<td>1376152</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gaymar Heating/Cooling</td>
			<td style="width:121px;">Braintree</td>
			<td style="width:161px;">TP-700</td>
			<td style="width:167px;">Heating Pad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass pipette puller</td>
			<td>Sutter Instrument,</td>
			<td style="width:161px;">P-97</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass pipettes</td>
			<td>Sutter Instrument,</td>
			<td>BF150-117-10</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Hair Removal Lotion</td>
			<td style="width:121px;">Nair</td>
			<td style="width:161px;">N/A</td>
			<td>Hair removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hartman Hemostats</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">13002-10</td>
			<td>IUE</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Open field maze- homemade acrylic arena</td>
			<td style="width:121px;">Custom-built</td>
			<td style="width:161px;">N/A</td>
			<td>50 &#215; 50 &#215; 30 cm length-width-height</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">pCAG-GFP</td>
			<td style="width:121px;">Addgene</td>
			<td>11150</td>
			<td>Mammalian expression vector for expression of GFP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Picospritzer III</td>
			<td>Parker Hannifin</td>
			<td style="width:161px;">N/A</td>
			<td>pressure injector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retractor - 2 Pronged Blunt</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">17023-13</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ring forceps</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">11103-09</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sterilizer, dry bead</td>
			<td style="width:121px;">Sigma</td>
			<td>Z378569</td>
			<td>sterelize surgical tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SUTURE, 3/0 PGA, FS-2, VIOLET FOR VET USE ONLY</td>
			<td>Havel&#39;s</td>
			<td>HJ398</td>
			<td>Suture muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Water bath</td>
			<td style="width:121px;">Cole-Parmer</td>
			<td>EW-12105-84</td>
			<td style="width:167px;">warming sterile saline</td>
		</tr>
	</tbody>
</table>

a pipeline using bilateral in utero electroporation to interrogate genetic influences on rodent behavior

ゲノムワイド関連研究は、多くの神経疾患の異種遺伝的基盤に光を当てるにつれて、脳の発達と機能に対する特定の遺伝子の寄与を研究する必要性が高まる。トランスジェニックマウスラインは非常に高価であり、多くの新しい疾患関連遺伝子はまだ市販の遺伝的線を持っていないため、特定の遺伝子操作の役割を研究するためにマウスモデルに頼ることは必ずしも実現可能ではありません。また、マウス ラインを作成するには、開発と検証に何年もかかる場合があります。子宮内エレクトロポレーションでは、特定の遺伝子操作を達成するためにDNAプラスミドを開発するだけで必要な生体内での細胞型特異的な方法で遺伝子発現を操作する比較的迅速かつ容易な方法を提供する。子宮内の両側性のエレクトロポレーションは、前頭皮錐体錐神経細胞の大規模な集団を標的にするために使用することができる。この遺伝子導入法と行動アプローチを組み合わせることで、前頭前野ネットワークの機能や若年マウスおよび成体マウスの社会的行動に対する遺伝子操作の効果を研究することができます。

カスタムエレクトロポレーターと3つのプロング電極の開発と実装に成功しました。 IUE の場合、前述の設計27に基づいて安価なカスタムエレクトロポレーターが構築されました (図 1Aおよび図 2)。突起の先端に2つの負極を取り付けたプラスチック鉗子を用いて23,24の突起?...

Watch this Scientific Journal Video about A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior at JoVE.com

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

神経精神疾患の病因における最近発見された疾患関連遺伝子の役割は依然として不明瞭である。子宮エレクトロポレーション技術における修飾された両側性は、ニューロンの大規模集団における遺伝子導入と、社会的行動に対する遺伝子発現変化の原因の影響の検討を可能にする。

子宮内電散法における両側性を用いたパイプラインによるげっ歯類の行動に対する遺伝的影響の調査

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...

a-pipeline-using-bilateral-utero-electroporation-to-interrogate

Research

JoVE Journal

Biology

3.9K ビュー.  Boston University. 神経精神疾患の病因における最近発見された疾患関連遺伝子の役割は依然として不明瞭である。子宮エレクトロポレーション技術における修飾された両側性は、ニューロンの大規模集団における遺伝子導入と、社会的行動に対する遺伝子発現変化の原因の影響の検討を可能にする。

ビデオ: A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

E16ラット胚の子宮内での脳室内注入とエレクトロポレーションで

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

生物学

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

E15マウス胚の子宮内での脳室内注入とエレクトロポレーションで

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

初期のニワトリ胚をex vivo（新文化）の文化のための方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

初期のニワトリ胚のin situハイブリダイゼーションダブルホールマウント

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

チックの全マウントの抗体染色のための方法

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

鶏胚における神経誘導のためのアッセイ

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

マウス網膜神経節細胞における遺伝子発現のための子宮内および生体外でのエレクトロポレーションで

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人間のDNA修復タンパク質の遺伝的研究は、モデルシステムとして酵母を使用

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

外酸生産の測定と解析は、解糖速度を決定するために

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...