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><tbody><tr><td>13mm Silk Black Braided Suture</td><td>Havel&#039;s</td><td>SB77D</td><td>Suture skin</td></tr><tr><td>Adson Forceps</td><td>F.S.T.</td><td>11006-12</td><td>IUE</td></tr><tr><td>C270 Webcam</td><td>Logitech</td><td>N/A</td><td>Record behavior</td></tr><tr><td>Electroporator</td><td>Custom-built</td><td>N/A</td><td>See Figure 1 and 2 and Bullmann et al, 2015</td></tr><tr><td>EZ-500 Spin Column Plasmid DNA Maxi-preps Kit, 20preps</td><td>Bio Basic Inc.</td><td>BS466</td><td>Pladmid preparation</td></tr><tr><td>Fast Green FCF</td><td>Sigma</td><td>F7252-5G</td><td>Dye for DNA solution</td></tr><tr><td>Fine scissors- sharp</td><td>F.S.T.</td><td>14060-09</td><td>IUE</td></tr><tr><td>Fisherbrand Gauze Sponges</td><td>Fisher Scientific</td><td>1376152</td><td>IUE</td></tr><tr><td>Gaymar Heating/Cooling</td><td>Braintree</td><td>TP-700</td><td>Heating Pad</td></tr><tr><td>Glass pipette puller</td><td>Sutter Instrument,</td><td>P-97</td><td>IUE</td></tr><tr><td>Glass pipettes</td><td>Sutter Instrument,</td><td>BF150-117-10</td><td>IUE</td></tr><tr><td>Hair Removal Lotion</td><td>Nair</td><td>N/A</td><td>Hair removal</td></tr><tr><td>Hartman Hemostats</td><td>F.S.T.</td><td>13002-10</td><td>IUE</td></tr><tr><td>Open field maze- homemade acrylic arena</td><td>Custom-built</td><td>N/A</td><td>50 &amp;times; 50 &amp;times; 30 cm length-width-height</td></tr><tr><td>pCAG-GFP</td><td>Addgene</td><td>11150</td><td>Mammalian expression vector for expression of GFP</td></tr><tr><td>Picospritzer III</td><td>Parker Hannifin</td><td>N/A</td><td>pressure injector</td></tr><tr><td>Retractor - 2 Pronged Blunt</td><td>F.S.T.</td><td>17023-13</td><td>IUE</td></tr><tr><td>Ring forceps</td><td>F.S.T.</td><td>11103-09</td><td>IUE</td></tr><tr><td>Sterilizer, dry bead</td><td>Sigma</td><td>Z378569</td><td>sterelize surgical tools</td></tr><tr><td>SUTURE, 3/0 PGA, FS-2, VIOLET FOR VET USE ONLY</td><td>Havel&#039;s</td><td>HJ398</td><td>Suture muscle</td></tr><tr><td>Water bath</td><td>Cole-Parmer</td><td>EW-12105-84</td><td>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

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4.0K 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 ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

사용하여 여러 개의 CNS 준주로 효율적인 유전자 전달 Utero에서 Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

신경과학

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 ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

몸에 붙이기 형질 전환 쥐의 발생에 의해<em&gt; 자궁 내</em&gt; 일렉트로 및<em&gt; 생체 내</em&gt; 생물 발광 상영

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

마우스의 유전자 조작을 통해 시상 하부 개발<em&gt; 자궁 내</em&gt; 일렉트로

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

배열 CGH, 전체 exome 시퀀싱과 설치류에 Utero에서 Electroporation 뇌 기형에 대 한 원인 유전자를 식별 하는 새로운 전략

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

자궁 내 일렉트로는 신경 세포의 부분 집단의 흥분 및 단일 세포의 연결을 연구에 접근

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

자궁에서 두 배의 자궁 전기화는 시간적으로 그리고 공간적으로 분리된 세포 인구를 표적으로 합니다

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although electroporation has traditionally been used in in vitro studies, scientific advancements have now broadened its utilization to intact organs, such as those found in mouse embryos developing in utero. This video will introduce the key principles behind in utero electroporation in addition to reviewing the basic surgical techniques required to access developing embryos within a pregnant rodent. Details of the injection and electroporation steps are provided along with important considerations for directing gene delivery to specific brain regions. Finally, neurobiological applications of in utero electroporation are presented, such as investigating how specific genes contribute to neural development and how connections form between developing neurons.

Murine In Utero Electroporation

쥐 In Utero 전기 천공

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and ...

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

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