우리는 중요한 피드백과 원고에 편집 리사 크레츠지 감사합니다. 우리는 행동 두뇌의 관류 및 세포 계산을 돕는 데 귀중한 크루즈 - 마르틴 실험실의 모든 연구 조수에게 감사드립니다. 우리는 삼극성 전극의 디자인에 대한 입력 안드레제이 Cwetsch 감사, 그리고 토드 블루테와 보스턴 대학 생물학 이미징 코어는 공초점 현미경의 사용에 대한. 이 작품은 NARSAD 젊은 조사자 그랜트 (AC-M, #27202), 브렌튼 R. 루츠 상 (ALC), I. 알덴 맥치 상 (ALC), NSF NRT UtB : 신경 광장 국가 연구 펠로우십 (ALC, #DGE1633516), 보스턴 대학 학부 연구 기회 프로그램 (WWY)에 의해 지원되었다. 기금은 연구 설계, 데이터 수집 및 분석, 출판 결정 또는 원고 준비에 아무런 역할이 없었습니다.

<ol>
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저자는 경쟁 이익을 선언하지 않습니다.

본명, 파이프라인은 전두엽 피질 뉴런의 큰 집단에 대한 관심의 새로운 유전자의 조작을 마우스의 행동 분석과 결합하는 것으로 기술된다. 더욱이, 이 파이프라인은 초기 산후 발달 과 성인기에 둘 다 동일 마우스에 있는 행동의 종방향 연구 결과를 허용합니다. 이 기술은 시간과 비용의 관점에서 비용이 많이 들 수 있는 유전 동물 모델에 의지할 필요성을 우회합니다. 이 프로토콜의 강도는 최근 ...

게놈 전체 협회 연구(GWAS)는 뇌 병리1,2,3,4와관련된 새로운 후보 유전자의발견을주도했다. 이러한 연구는 정신 분열증 (SCZ)과 같은 치명적인 신경 정신 장애를 이해하는 데 특히 도움이되었으며, 새로운 유전자의 조사는 새로운 연구 및 치료 개입 라인의 시작 점으로 사용되었습니다5,6. SCZ에 대한 위험을 품고 있는 유전자는 산전 및 조기 산후 발달 중 전두엽 피질(PFC)에서 편향된 발현을 나타내며, 이는 여러 신경정신장애의 병리학에 연루된영역(7)이다. 또한, 정신 장애의 마우스 모델은 PFC 네트워크6,8,9에서비정상적인활성을나타낸다. 이 결과는 SZC 관련 유전자가 이 지역의 발달 배선에 있는 역할을 할 지도 모른다는 것을 건의합니다. 동물 모델을 이용한 추가 조사는 PFC에 있는 연결의 설치에 이 후보 유전자의 기여를 이해하고 이 유전자가 신경 정신병 장애의 병인에 있는 원인 역할이 있는지 결정하기 위하여 요구됩니다. 산전 및 조기 산후 발달 중 특정 뉴런 회로에 대한 유전자 발현 변화를 연구할 수 있는 마우스의 유전자 조작 기술은 유전자 발현 변화를 PFC 기능 장애에 연결하는 분자 메커니즘을 이해하는 유망한 방법이다.
유전 마우스 라인은 뇌 발달과 기능에 특정 유전자의 영향을 연구하는 방법을 제공합니다. 그러나, 형질 전환 쥐에 의존하는 것은 신경 회로 개발에 특정 유전자의 효력을 검토하기 위하여 항상 상업적으로 이용 가능한 라인이 없기 때문에 제한될 수 있습니다. 또한 사용자 지정 마우스 라인을 개발하는 데 매우 비용이 많이 들고 시간이 많이 소요될 수 있습니다. 형질전환 마우스와 바이러스 접근법을 결합한 교차 유전자 조작 전략의 사용은 뇌10,11,12의 이해에 혁명을일으켰습니다. 많은 진보에도 불구하고, 바이러스 전략은 바이러스발현(13)과 바이러스 발현(14)과 관련된 세포 독성을제한할 수 있는 포장 용량의 한계를 포함하여 바이러스 벡터 유형에 따라 일정한 한계를 가지고 있다. 더욱이, 대부분의 실험 조건에서, 아데노 관련 바이러스를 이용한 강력한 유전자 발현(AAV)은 약 2~4주15일이필요하며, 조기 산후 발달 시 유전자를 조작할 수 없는 일상적인 바이러스 전략을 만드는 것이다.
자궁 전기기화(IUE)에서 신속하고 저렴한 유전자 전달을 허용하는 대체 접근법은형광 라벨링 및 약리유전학 적 또는 광유전학적 접근법과 결합될 때, 뉴런 회로의 기능을 해부하는 강력한 플랫폼을 제공한다. 또한, CRISPR-Cas9 게놈 편집 유전자의 발달과 함께 특정 유전자의 세포형 특이적 노크다운 또는 노크아웃을 통해 또는프로모터(18,19)의변조를 통해 과발성 또는 정확하게 변경될 수 있다. IUE를 이용한 유전자 조작 접근법은 유전자가 좁은 발달창(20)동안 시험될 필요가 있을 때 특히 유리하다. IUE는 다목적 기술이며 특정 프로모터 하에서 발현 벡터에 유전자를 삽입하여 쉽게 수행될 수 있다. 유전자 발현의 추가 제어는 상이한 강점의 프로모터를 이용하여 발현을 구동하거나 유전자발현(21,22)을일시적으로 조절할 수 있는 유도성 프로모터를 사용하여 달성될 수 있다. 추가적으로, IUE는 특정 피질 층, 세포 모형 및 두뇌 지구 내의 세포의 표적을 허용합니다, 다른 접근을 사용하여 항상 가능하지 는 않습니다5,17. 보다 효율적인 전극 분포를 생성하는 3개의 전극의 사용에 기초한 IUE 구성의 최근 발전은 이 방법의 기능적 레퍼토리를 확대하고 과학자들이 새로운 세포 유형을 표적으로 하고23,24를표적으로 할 수 있는 세포의 효율, 정확성 및 수를 증가시킬 수 있게 하였다. 이 기술은 최근 PFC 기능 및 초기 인식5에서SCZ에 연결된 유전자인 보완 성분 4A(C4A)의 원인 역할을 결정하는 데 사용되었다. 
여기에 제시된 유전자 전송 접근은 PFC를 포함하여 전두엽 피질에 있는 흥분성 뉴런의 큰 인구를 표적으로 하는 유전자 전송 접근을 결합하는 실험 적인 파이프라인입니다, 세포 및 회로 수준 변경의 연구 결과를 가능하게 하는 행동 패러다임, 그러나 또한 초기 산후 발달 및 성인기에 걸쳐 행동을 감시할 수 있습니다. 먼저 설명된 방법은 정면 피질 영역에서 층(L) 2/3 피라미드 뉴런의 큰 집단을 양자간 횡단하는 방법이다. 다음으로, 청소년과 성인 마우스의 사회적 행동을 분석하는 작업이 설명되어 있습니다. 세포 수는 세포 형질의 범위와 위치를 정량화하기 위해 행동 작업이 완료되면 얻을 수 있습니다. 더욱이, 전형된 세포의 수는 행동 데이터와 상관관계가 있을 수 있고, 더 많은 수의 전과세포가 행동에 더 큰 혼란을 초래하는지 확인할 수 있다.

<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

게놈 전체 협회 연구가 많은 신경 질환의 이기종 유전 기초에 빛을 발산으로, 뇌 개발 및 기능에 특정 유전자의 기여를 연구 할 필요가 증가. 생생 마우스 라인이 매우 비용이 많이 들고 많은 새로운 질병 관련 유전자가 아직 상업적으로 이용 가능한 유전 라인을 가지고 있지 않기 때문에 특정 유전 조작의 역할을 연구하기 위하여 마우스 모형에 의지하는 것은 항상 가능하지 않습니다. 또한 마우스 선을 만드는 데 수년간의 개발 및 유효성 검사가 걸릴 수 있습니다. 자궁에서 전기 기공은 특정 유전 적 조작을 달성하기 위해 DNA 플라스미드를 개발해야만 하는 생체 내에서 세포 형 특정 방식으로 유전자 발현을 조작하는 비교적 빠르고 쉬운 방법을 제공한다. 자궁 전기화의 양자는 전두엽 피질 피라미드 뉴런의 큰 인구를 표적으로 하기 위하여 이용될 수 있습니다. 이 유전자 전달 방법과 행동 접근법을 결합하면 전두엽 피질 네트워크의 기능과 청소년 및 성인 마우스의 사회적 행동에 대한 유전 조작의 효과를 연구 할 수 있습니다.

맞춤형 전기포레이터와 3개의 갈래 전극의 성공적인 개발 및 구현. IUEs의 경우, 이전에 설명된설계(27)를 기반으로 저렴한 맞춤형 내장 전기포레이터를 제작하였다(도1A 및 도 2). 3개의 갈래 전극이23,24개의 플라스틱 집게를 사용하여 갈래의 끝에 부착된 2개의 음극을 사용했고...

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

신경 정신 장애의 병인에 있는 최근에 발견된 질병 관련 유전자의 역할은 모호하게 남아 있습니다. 자궁 전기 기화 기법에서 수정된 양자간은 뉴런의 큰 인구에서 유전자 전송을 허용하고 사회적 행동에 대한 유전자 발현 변화의 원인 효과의 검사를 허용한다.

자궁 전자기화에서 양자 간을 사용하여 설치류 행동에 대한 유전적 영향을 심문하는 파이프라인

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 조회수.  Boston University. 신경 정신 장애의 병인에 있는 최근에 발견된 질병 관련 유전자의 역할은 모호하게 남아 있습니다. 자궁 전기 기화 기법에서 수정된 양자간은 뉴런의 큰 인구에서 유전자 전송을 허용하고 사회적 행동에 대한 유전자 발현 변화의 원인 효과의 검사를 허용한다.

비디오: 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 랫 태아의 Utero의 심실 주입 및 Electroporation에

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

연구

생물학

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 마우스 태아의 Utero의 심실 주입 및 Electroporation에

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.

조기 칙 태아 예 생체내 (새로운 문화)의 문화에 대한 방법

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.

조기 칙 태아의 현장 하이브리드화에서 더블 전체 산

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.

칙의 전체 마운트 항체 스테 이닝을위한 방법

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.

칙의 배아에서 신경 유도에 대한 분석

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.

마우스 망막 신경절 세포의 유전자 발현에 대한 utero 및 전직 생체내의 Electroporation에

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.

인간의 DNA 수리 단백질의 유전자 연구는 모델 시스템으로 효모를 사용하여

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.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

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.

세포 외 산 생산의 측정 및 분석은 당분 속도를 확인하는

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