Name: structure-function studies in mouse embryonic stem cells using recombinase-mediated cassette exchange
Uploaded: 2017-04-27T15:00:00
Description: Watch this Scientific Journal Video about Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange at JoVE.com

우리는 우수한 기술 지원을 Jinke D&#39; Hont, 프레 더 리크 반 Rockeghem, 나탈리 Farla, 켈리 레메어 및 Riet 드 RYCKE 감사합니다. 우리는 또한 전문가의 도움에 대한 염증 연구 센터의 Bioimaging 핵심 시설에서 EEF Parthoens, Evelien 반 Hamme, 아만다 Goncalves은 감사합니다. 우리는 가치있는 토론에 대한 우리의 연구 그룹의 구성원을 인정합니다. 이 작품은 벨기에의 과학 정책에 의해 지원되었다 (Belspo Interuniversity 명소 폴란드 - 상 IAP VII-07 DevRepair, https://devrepair.be) 퀸 엘리자베스 의료 재단, 벨기에 (GSKE 2,008에서 2,010 사이에 의해;에 http : // www가 ) - gske.be을 .fmre하고, 겐트 대학, 벨기에 (http://www.ugent.be/en/ghentuniv)의 공동의 연구 작업 (GOA 01G01908)에 의해. SG는 플랑드르 연구 기금 (FWO-V)의 박사후 연구원이다.

마우스에 대한 모든 실험은, 기관, 국가 및 유럽의 동물 규정에 따라 실시 하였다. RMCE 호환 KO의 mESCs 1. 분리 <ol><li> 이러한 ROSALUC 마우스 10 ROSA26-IPSC 마우스 (21) 등 RMCE 호환 마우스와 이형 KO 쥐 품종. 두 RMCE 호환 마우스는 혼합 129 / C57BL6 / 스위스 배경에 유지되었다. 참고 : 이형 KO 마우스를 교차은 동형 접합 KO 마우스의 배아 치사를 극복하는 것이 ?...

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우리 MESC 분리 방법은 사용하기 쉬운 및 배반포의 미세 같은 고급 기술이나 장비를 필요로하지 않습니다. 따라서,이 기술은 과학계의 큰 비율에 액세스 할 수 있습니다. 기본 세포 배양 경험을 가진 사람은 ICM의의 outgrowths을 전파하고 mESCs 라인을 설정할 수 있습니다. 그러나 배반포의 세척 및 처리는 연습이 필요합니다. 구강 내에 피펫 포배를 전송하는 데 사용되는 마이크로 피펫, 마이크로 피펫 ...

포유류의 게놈은 약 20,000 단백질 코딩 유전자를 포함하는 것으로 추정된다. 스 플라이 싱 및 번역 후 변형은 상기 단백질 레퍼토리를 증가시킨다. 단백질은 모듈 식 구조 (1)을 가지고 있고 종종 서로 다른 단백질 복합체에 자신의 모집 및 여러 세포 과정 2에 참여를 허용 여러 상호 작용 도메인을 포함한다. 한 가지 예는 p120ctn라는 다기능 단백질이다. p120ctn Ctnnd1는 유전자에 의해 코딩 및 N 말단 및 C 말단 영역에 의해 측면 큰 중앙 딜 반복 영역으로 구성된다. p120ctn의 딜 도메인이 세포 - 세포 부착에 관여하는 고전 cadherins의 고도로 보존 된 막 근접 영역에 결합 할뿐만 아니라, 전사 리프레 가이 소 (海)에 결합한다. p120ctn의 N- 말단 도메인은 다른 키나제, 포스파타제, 작은 RhoGTPases, 및 미세 소관 - 연관된 페이지와 상호 작용roteins 3. 흥미롭게도, 스 플라이 싱의 결과, p120ctn 이성체 네 대체 개시 코돈 (4)로부터 생성 될 수있다. 이 개시 코돈 &#39;가장 -5- 번역 및 전장 N 말단 부분을 포함한다으로 p120ctn 이성체 (1A)는, 최장. p120ctn 3 및도 4는,이 N 말단 부분을 부분적으로 완전 각각 삭제 이소 폼. 다른 세포 기능의 단백질 (또는 단백질 이소 형)과 도메인의 정확한 역할을 이해하는 것은 과제로 남아. mESCs 타겟팅 유전자는 해당 유전자의 유전 삭제를 통해 단백질의 기능 연구를 가능하게하고 널리 발달 중요한 질병 관련 유전자 및 경로의 식별에 기여하고있다. 역 유전학이 침투 의한 상동 재조합 5 MESC 분리 및 표적 유전자의 분야에서의 진보의 결과였다 </s&gt;까지. 상동 재조합은 DNA 단편은 이중 가닥 (DS) DNA 휴식 후 2 개 유사하거나 동일한 핵산 잔기 사이에서 교환되는 과정이다. dsDNA 나누기가 자주 있기 때문에 일반적으로, HR은 비효율적이다. 최근 타겟팅 상동 관한 유전자의 효율이 부위 특이 적 뉴 클레아 제 (6, 7)를 사용하여 증가 될 수 있지만, 불행하게도, 이러한 오프 - 표적 효과 8 쉽다. 보다 신뢰성있는 기술은 표적 유전자를 활성화하는 등 Cre 호텔 /은 loxP 또는 FLPe / FRT 등의 부위 특이 적 재조합 시스템에 근거 RMCE이다. 및 FRT에 loxP 서열은 P1 박테리오파지에서 발견 사카 cerevisiae에 각각, 상기 위치의 방향을 결정하는 비대칭 8 염기쌍 서열을 포함하여 34 염기쌍으로 구성. 반면에,의 방향은 예를 들면, DNA 스트레치 내 두에 loxP 사이트는 floxed DNA 절제된다 여부를 확인 아니면 내가 것Cre 호텔 - 중재 재조합 9시 nversed. 이 개 사이트는 서로 다른 염색체에있는 경우 또한, Cre 호텔도 전좌를 유도 할 수 있습니다. RMCE 교차 반응하지 않고 그 게놈 궤적에 포함 된 이종 재조합 사이트 활용합니다. 같은 이종 사이트 어귀 DNA 단편을 포함하는 도너 플라스미드의 존재에서, 재조합으로 인해 이중 동시 전위 (도 1)의 RMCE 호환 게놈 로커스에이 DNA 단편을 삽입하는 것이다. 여기에만 정확하게 RMCE 타겟 클론 복원 수신 벡터상의 프로모터에 약제 내성 덕분에 렌더링 할 수있는 &quot;포획&quot;프로모터 이하 네오 마이신 내성 유전자 도킹 세포 R26 게놈에 존재하는 (네오 R) (도 1) 10, 11. 이 &lt;100 % 11 종종 가까운 매우 높은 타겟팅 효율성, 결과/ SUP&gt; 12. 결론적 RMCE 기반 타겟팅 매우 효율적인 구조 및 기능 연구를 위해 사용될 수있다; 그러나, 사전 설계 게놈 궤적을 필요로한다. <img alt="그림 1" src="/files/ftp_upload/55575/55575fig1.jpg" /> RMCE 매개 타겟팅 그림 1. 도식 표현. 모두 두 이종 FRT 부위 (흰색과 빨간색 삼각형으로 도시)을 품고있는 경우 RMCE 정의 된 게놈 로커스에 들어오는 대상 벡터에서 DNA 세그먼트의 교환을 허용한다. 또한, 유전자 조작 궤적이 promoterless 및 절단 네오 마이신 내성 (네오 R) 유전자를 포함한다. 들어오는 DNA 단편의 코돈 프로모터를 제공하고 시작함으로써 만 정확한 재조합 높은 타겟팅 효율 결과, 네오 마이신 저항성을 복원. <a href="http://ecsource.jove.com/files/ftp_upload/55575/55575fig1large.jpg" target="_blank">t의 더 큰 버전을 보려면 여기를 클릭하십시오</a>그의 그림. mESCs에서 게놈 엔지니어링은 RMCE 호환 마우스의 생성을 허용합니다. 1981 년에, 두 그룹은 배반포의 내부 세포 덩어리 (ICM)로부터 다 능성 세포를 포착 및 배양 13, 14에서 그 유지에 성공했다. mESCs는 세균 세포 계보를 포함하여 배아 및 성체 세포의 모든 유형에자가 재생과 분화 할 수있다. 따라서 mESCs 타겟팅 유전자는 구조적 또는 (정속 신장식 /에 loxP 시스템을 이용하여) 조건부 KO 마우스의 개발을 통해 역 유전 연구를 가능하게한다. 그러나, 마우스 ES 세포를 분리하는 고전적인 방법은 매우 비효율적이다. 몇몇 주요 개선은 매우 SR 및 소 태아 혈청 (FBS) (16)를 포함 MESC 매체 번갈아 15 배지 정의 혈청 여분 (SR)의 사용을 포함하여, 유도 MESC 라인 성공률을 증가 및 약리학 사용했다이러한 pluripotin 또는 2I 17 논리 화합물. Pluripotin 작은 합성 분자, 백혈병 억제 인자 (LIF) 및 마우스 배아 섬유 아 세포 (MEFs) (18)의 부재 하에서 미분화 상태 mESCs의 전달을 허용한다. 마지막으로,이 mESCs는 SR / FBS 배지 교대 프로토콜 (20)과 함께 LIF 및 19 pluripotin 결합 될 때 (100 %에 가까운) 아주 높은 효율로 분리 될 수 있음을 보여왔다. 이러한 프로토콜은 이후 구조 기능 연구에 사용할 수 있습니다 RMCE 호환 KO의 mESCs의 효율적인 분리를 할 수 있습니다. 이 논문은 하나의 특정 세포 과정을 담당하는 단백질 내에서 키 도메인 또는 잔류 물을 식별 할 수있는 방법을 설명합니다. 이를 위해, 효율적인 MESC 분리를 가능하게 고급 기술, 대상 벡터 조립 및 MESC 타겟팅의 파이프 라인을 생성했다디. 단백질 이성체, 도메인 돌연변이 및 다운 스트림 이펙터와 같은 대형 패널은 KO의 mESCs에 도입 될 수 있으며, 체외 KO 표현형을 구출 할 수있는 능력을 평가 할 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="766">
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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">ROSALUC Mice</td>
			<td style="width:143px;">made in house</td>
			<td style="width:107px;"></td>
			<td style="width:245px;">frozen sperm available upon request</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">R26-iPSC mice</td>
			<td style="width:143px;">made in house</td>
			<td style="width:107px;"></td>
			<td style="width:245px;">frozen sperm available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Vessel probe</td>
			<td style="width:143px;">Fine Science Tools</td>
			<td>10160-13</td>
			<td style="width:245px;">to check for copulation plugs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">M2 medium</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">M7167</td>
			<td style="width:245px;">make aliquots and store at -20&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Fine forceps (Dumont #5 Standard tip Student forceps)</td>
			<td style="width:143px;">Fine Science Tools</td>
			<td style="width:107px;">11251-10</td>
			<td style="width:245px;">spray with 70% EtOH before use (do not autoclave)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">23G needles</td>
			<td style="width:143px;">Fine-ject</td>
			<td style="width:107px;">8697</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">1-ml syringes</td>
			<td style="width:143px;">Soft-ject</td>
			<td style="width:107px;">6680</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">60-mm bacterial grade plates (for flushing)&#160;</td>
			<td style="width:143px;">Gosselin</td>
			<td style="width:107px;">BB60-01</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Mouth pipette</td>
			<td style="width:143px;">made in house</td>
			<td style="width:107px;"></td>
			<td style="width:245px;">see discussion</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Mouse embryonic fibroblasts (MEFs, TgN (DR4)1 Jae strain)</td>
			<td style="width:143px;">ATTC</td>
			<td style="width:107px;">SCRC-1045</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">TgN (DR4)1 Jae mice</td>
			<td style="width:143px;">The Jackson Laboratory</td>
			<td style="width:107px;">3208</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Mitomycin C&#160;</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">M0503</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Phosphate buffered saline (PBS) without calcium or magnesium</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">14190-094</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:272px;">Tg(DR4)1Jae/J mice</td>
			<td style="width:143px;">JAX</td>
			<td style="width:107px;">3208</td>
			<td style="width:245px;">mice that contain four drug-selectable genes and DR4 MEFS confers resistance to neomycin, puromycin, hygromycin and 6-thioguanine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">0.1% Gelatin</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">G1393</td>
			<td style="width:245px;">Dissolve 0.5 g in 500 ml distilled water, autoclave and store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Trypsin (0.25%)&#160;</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">25200-056</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">2 &#956;M pluripotin</td>
			<td style="width:143px;">Cayman Chemical</td>
			<td style="width:107px;">10009557</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:272px;">pRMCE-DV1</td>
			<td style="width:143px;">BCCM/LMBP collection&#160;</td>
			<td style="width:107px;">LMBP 08870</td>
			<td style="width:245px;">public available from the BCCM/LMBP collection (http://bccm.belspo.be)&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:272px;">cre-excised pRMCE-DV1</td>
			<td style="width:143px;">BCCM/LMBP collection&#160;</td>
			<td style="width:107px;">LMBP 08195</td>
			<td style="width:245px;">public available from the BCCM/LMBP collection (http://bccm.belspo.be)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">pCAG-FlpE-IRES-Puro-pA&#160;</td>
			<td style="width:143px;">Addgene</td>
			<td style="width:107px;">20733</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">heat-shock competent DH5&#945; bacteria&#160;</td>
			<td style="width:143px;">made in house</td>
			<td style="width:107px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Gateway pDONR221 vector</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">12536-017</td>
			<td style="width:245px;">contains kanamycin-resistance gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BP clonase II mix</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">11789-020</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">LR clonase II mix</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">11791-020&#160;</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Luria Broth (LB)&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Ampicillin&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Applied Biosystems 3730XL DNA Analyzer</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">3730XL</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">G418</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">11811-023</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Lipofectamine 2000 transfection reagent</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">11668027</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Lipofectamine LTX transfection reagent</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">15338100</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Effectene transfection reagent</td>
			<td style="width:143px;">Qiagen</td>
			<td style="width:107px;">301425</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GATEWAY pENTR 1A vector</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">A10462</td>
			<td>recombination-compatible vector</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mouse monoclonal anti-p120ctn antibody</td>
			<td style="width:143px;">BD Transduction Laboratories</td>
			<td style="width:107px;">610134</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mouse monoclonal anti-Ecadherin antibody</td>
			<td style="width:143px;">BD Transduction Laboratories</td>
			<td style="width:107px;">610181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">General equipment:</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Sterile dissection tools</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;">fine scissors and forceps for dissecting the uterus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sterile pipettes: 5 ml, 10 ml and 25 ml</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">15-ml and 50-ml conical centrifuge tubes</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:272px;">96-well culture plates V-shaped bottom and flat bottom)&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Culture dishes: 24 wells, 12 wells and 6 wells</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Multichannel pipettes (to pipette 30, 50, 100 and 200 &#956;l)&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sterile multichannel reservoirs</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Access to a laminar air flow</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:272px;">Access to an incubator at 37&#176;C with 5% CO2</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Access to an inverted microscope</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Access to a bench-top centrifuge</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Access to a stereo microscope with transmitted-light&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture media:</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MEF Medium:</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;">stored at 4&#176;C; warm 30 min at 37&#176;C before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">&#160;Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">41965-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">10% fetal bovine serum (FBS)</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">F-7524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">L-glutamine (2 mM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">25030-024&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sodium pyruvate&#160; (0.4 mM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">11360-039&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">penicillin (100 U/ml)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">15140-122&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">streptomycin (100 &#181;g/ml)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">15140-122&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">SR-based mESC medium:</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;">stored at 4&#176;C; warm 30 min at 37&#176;C before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">DMEM/F12</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">31330-038</td>
			<td style="width:245px;">mixed in a 1:1 ratio</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">15% knock-out serum replacement (SR )</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">10828&#8211;028</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">L-glutamine (2 mM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">25030-024&#160;</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">0.1 mM non-essential amino acids&#160;</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">11140-050</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">penicillin (100 U/ml)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">15140-122&#160;</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">streptomycin (100 &#181;g/ml)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">15140-122&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">&#946;-mercaptoethanol (0.1 mM)&#160;</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">M 3148&#160;</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">2,000 U/ml recombinant mouse LIF&#160;</td>
			<td style="width:143px;">(IRC/VIB Protein Service facility)</td>
			<td style="width:107px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">FBS-based mESC medium (similar to SR-based mESC medium):</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;">stored at 4&#176;C; warm 30 min at 37&#176;C before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Knockout DMEM</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">10829-018&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">15% FBS</td>
			<td style="width:143px;">Hyclone</td>
			<td style="width:107px;">SH30070.03E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Differention Medium</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;">stored at 4&#176;C; warm 30 min at 37&#176;C before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">21980-032&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">15% FBS</td>
			<td style="width:143px;">Hyclone</td>
			<td style="width:107px;">SH30070.03E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">5% CD Hybridoma Medium(1x) liquid&#160;&#160;</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">11279-023&#160;</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">&#160;2 mM L-glutamine</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">25030-024&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">0.4 mM 1-thioglycerol</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">M-6145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">50 &#956;g/ml ascorbic acid</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">A-4544&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">penicillin (100 U/ml)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">15140-122&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">streptomycin (100 &#181;g/ml)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">15140-122&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2i</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">1 &#956;M Erk inhibitor PD0325901&#160;</td>
			<td style="width:143px;">Axon Medchem</td>
			<td style="width:107px;">Axon 1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">3 &#956;M Gsk3 inhibitor CHIR99021</td>
			<td style="width:143px;">Axon Medchem</td>
			<td style="width:107px;">Axon 1386</td>
			<td></td>
		</tr>
	</tbody>
</table>

structure-function studies in mouse embryonic stem cells using recombinase-mediated cassette exchange

마우스 배아 또는 배아 줄기 세포 (mESCs)에서 유전자 공학은 주어진 단백질의 기능 연구를 위해 수 있습니다. 단백질은 세포 골격을 이루는 종종 번역 후 변형에 의해 영향을받을 수있는 다수의 기능 영역들로 구성된다. 조건부 또는 구성 적 녹아웃 (KO) 마우스에서 전체 단백질의 고갈이 고려 기능적 다양성 및 규제를받지 않는다. MESC 라인 FLPe 재조합 매개 카세트 교환 (RMCE)을위한 도킹 사이트가 ROSA26 (R26)의 궤적 내에 삽입되는 유도 마우스 모델은 이전에보고되었다. 여기, 우리는 멀티 도메인 단백질의 서로 다른 기능의 분자 해부를 허용하는 구조 기능 접근에보고한다. 이를 위해, RMCE 호환 마우스는 KO 마우스를 교차해야하고 RMCE 호환 KO의 mESCs는 격리해야합니다. 다음으로, 추정 구조 구조의 패널의 RMCE targeti 통해 R26 로커스에 도입 될 수있다NG. 후보 구조 cDNA를 쉽게 재조합 복제를 사용하여 대상 벡터의 RMCE 사이트 사이에 삽입 할 수 있습니다. 다음에, 코 mESCs는 FLPe 재조합 발현 플라스미드와 함께 타겟팅 벡터로 형질 감염된다. RMCE는 ROSA26 도킹 사이트에서 프로모터 이하 네오 마이신 - 내성 유전자를 재 활성화하고 정확한 표적화 이벤트를 선택할 수 있습니다. 이러한 방식으로, 100 %에 가까운 높은 타겟팅 효율이 반 고 스루풋 추정 방식 다중 구조 구조의 삽입을 허용 얻어진다. 마지막으로, R26 기반 구조 구조의 다수는 부모 KO의 mESCs에서 관찰 된 표현형을 구출하기 위해 자신의 능력을 테스트 할 수 있습니다. 우리는 P120의 카테닌 (p120ctn) 표현형 판독으로 배아 체 (교환 사채)에서 내배엽 분화를 사용 KO의 mESCs의 원리 증명 구조 기능 연구를 제시한다. 이러한 접근 방식은 중요한 영역의 식별, 추정 다운 스트림 경로, 질병 관련 지점을 수 있습니다주어진 단백질 KO의 표현형의 기초가 돌연변이.

RMCE 호환 KO MESC 라인을 분리하는 절차는 그림 2에 도시되어있다. 두 연속 breedings는 RMCE 호환 KO의 배반포를 얻기 위해 필요합니다. 첫째, 이형 KO 마우스는 RMCE 호환, 이형 KO 마우스를 얻기 위해 RMCE 호환 마우스와 교차된다. 이러한 마우스는 RMCE 호환 3.5 DPC, 동형 KO의 포배를 얻었다 다른 이형 KO 마우스와 교배 시간 제한 사용된다. 멘델의 유전 법칙에 의해 예측 ?...

Watch this Scientific Journal Video about Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange at JoVE.com

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

단백질은 종종 다른 세포 기능을 발휘할 수있는 여러 도메인을 포함한다. 유전자 녹아웃 (KO)이 기능의 다양성을 고려하지 않는다. 여기, 우리는 다양한 기능 영역 또는 단백질의 변이의 분자 해부를 허용 KO 배아 줄기 세포에서 재조합 중재 카세트 교환 (RMCE) 기반 구조 - 기능 접근 방식을보고합니다.

재조합 효소 매개 카세트 교환을 사용하여 마우스 배아 줄기 세포의 구조 - 기능 연구

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

The overall goal of this structure-function method is to dissect at the molecular level the role of different protein domains, or disease-relevance mutations, in naive or differentiated mouse embryonic stem cells. This method can help to reveal how different residues or domains of a protein can contribute to its function in a given cellular context. The main advantage of this technique is that it allows very efficient targeting of rescue constructs to the genome of knock out embryonic stem cells, or ES cells, and the investigation of their role in different cell types.To achieve this, we combine three highly efficient technologies. These include improved mouse embryonic stem cell isolation, recombination-based targeting factor assembly, and ES cell targeting via recombination-mediated cassette exchange, or RMC. Demonstrating some of the procedures will be Jinke D'Hont, a technician from my laboratory.Recombination-mediated cassette exchange, or RMCE, allows the interchange of DNA fragments between a vector and a genomic locus. RMCE takes advantage of hetero-specific recombination sites that do not cross-react and that are embedded in a genomic locus. In the presence of a donor plasmid that contains a DNA fragment flanked by the same hetero-specific sites, the recombinase will insert this DNA fragment into the RMCE-compatible genomic locus because of double simultaneous translocation.Only upon correct recombination, the trapped promoterless Neomycin-resistance gene in the Rosa 26 docking site, will be restored via a PGK promoter in the incoming targeting vector, which renders drug-resistance. This Neomycin resistance trap system results in a very high targeting efficiency, often close to 100%The day before blastocyst isolation, add 0.1%gelatin, to 12 well-cultured plates. And incubate them at 37 degrees Celsius for five minutes.Aspirate the gelatin solution. Then seed one quarter of a vial of P2 Mouse Embryonic Fibroblasts, or MEFs, into MEF medium, and divide over the 12-well plate. Incubate the 12-well plates of MEFs in two milliliters of MEF medium at 37 degrees Celsius.And grow the cells to a confluent monolayer. Then add 10 micrograms per milliliter of Mitomycin seed to the wells. And incubate the cultures for three hours at 37 degrees Celsius, to inactivate the cells.After selecting heterozygous knock out mice containing an RMCE cassette in the Rosa 26 locus according to the text protocol, set up matings to breed RMCE-compatible heterozygous knock out mice, with heterozygous knock out mice. The following morning, check for copulation plugs by lifting the female by the base of the tale, and examine the vaginal opening for a whitish mass. If the plug is difficult to see, with an angled probe, slightly spread the lips of the vulva, then separate the plugged females from their males.To collect blastocysts at 3.5 days post-coitus, or DPC. After euthanizing the pregnant females, make a mid-ventral incision, and use fine scissors and forceps to dissect the uterus and oviduct. Next, bend a 26-gauge needle into a 45-degree angle attached to a 1 milliliter syringe filled with M2 medium.And insert the needle into the end of the uterus that is closest to the oviduct. Use fine forceps to hold the needle in place while pushing the plunger to flush the blastocysts from the uterus into the lid of a 10 centimeter dish. The uterus will swell, if the flushing is successful.Use a mouth pipette to collect all the embryos in a drop of M2 medium, and wash the embryos twice in a drop of M2 medium. Immediately after washing the embryos, use PBS, to wash the mitomycin-C inactivated cells twice. Then using a mouth pipette, plate each blastocyst onto a separate well of the mitomycin-C treated MEFs, in SRES cell medium, supplemented with pluripotent or 2I.Incubate the cultures at 37 degrees Celsius, and 5%carbon dioxide. Refresh the SREF cell medium every two to three days. Examine each blastocyst under a stereo microscope at 4X magnification, and check for hatching and detachment to the MEF layer.After 10 to 12 days of culture, under a stereo microscope, use a P10 pipette with disposable tips, to pick individual icium outgrowths. Transfer each outgrowth into approximately 10 microliters of medium to a V-shaped 96-well plate, containing 30 microliters per well of PBS. Using a multi-channel pipette, add 50 microliters of 0.25%trypsin to each well.And incubate the plate at 37 degrees Celsius and 5%carbon dioxide for three minutes. Add 100 microliters of pre-incubated FPS-containing ES cell medium, and dissociate the icium outgrowths into single cells by pipetting 10 to 15 times. Then transfer the dissociated cells to mitomycin-C treated 96 well MEF plates.The following day, change the SR-based medium. Expand the ES cells in a similar fashion from 24 to 6-well plates. After identifying rescued CDNA vectors according to the text protocol, prepare a 10 microliter LR reaction using 100 nanograms of rescue CDNA vector, 150 nanograms of pre-excised RMCEDV1 vector, and two microliters of recombinase mix, containing a phage-encoded integrase, an excisionase, and a bacterial integration host factor.Incubate the reaction at 25 degrees Celsius for two hours. To transform the recombined vectors, add 5 microliters of each mixture, to 40 microliters of heat shock competent E.Coli bacteria, in a 2 milliliter skirted screw cap tube. And incubate the sample on ice for 20 minutes.Then incubate the cells at 37 degrees Celsius for five minutes. Add one milliliter of LB medium to the tube, and incubate the cells at 37 degrees Celsius for one hour. Plate 50 microliters on agar plates with ampicillin.And grow at 37 degrees Celsius overnight. Identify colonies with the correct targeting vector by using a P200 tip to randomly pick five colonies. Transfer the tip to a glass test tube containing two to five milliliters of LB medium, and grow overnight at 37 degrees Celsius.After extracting the DNA from each colony, validate the samples by digesting 0.5 to two micrograms of DNA, and separate the samples on a 1%agarose gel. Start a culture of RMCE compatible knock out ES cells, and passage them at least twice on MEFs, in FBS-based ES cell medium. Then split the ES cells on a gelatinized six-well plate.The following day, use 1.5 milliliters of FBS-based ES cell medium to refresh the ES cells that are about 50%confluent. Combine one microgram of pre-excised RMCEDV1 targeting vector, containing rescue CDNA, and one microgram of FLIPe expression plasmid, in 250 microliters of pure DMEM medium. Add seven microliters of lipofectin-based transvection reagent to 250 microliters of pure DMEM medium.And incubate the solution for five minutes at room temperature. Combine the DNA and lipofectin solutions. And incubate the mixture at room temperature for 20 minutes.Then pipette the transvection mixture onto the refreshed ES cells, and gently swirl. One day after transvection, split all ES cells from the tube to a 10-centimeter culture dish, with a confluent layer of DR4 MEFS, and 10 milliliters of FPS-based ES cell medium. Two days after transvection, select ES cell clones with the correct FLIP-e mediated cassette exchange, by adding G418 to the medium.As shown here, massive killing of non-targeted colonies should be visible after three to five days. Colonies should appear after seven to 10 days. Pick these colonies, then expand them, and verify the clones as demonstrated earlier in this video.To differentiate ES cells into embryoid bodies, or EBs, after culturing knock out ES cells with Rosa 26-driven rescue constructs according to the text protocol, allow EBs to form in the non-adherent dishes for 30 days. Refresh the medium every two to three days by transferring the EB suspension to a 50 milliliter tube. And let the EBs settle by gravity.Remove the supernatant, add fresh medium, and transfer the EB suspension to a bacterial-grade dish. Analyze the ES cells and EBs by immunofluorescence and TEM, according to the text protocol. Using the structure function method, five pre-excised RMCEDV1 targeting vectors were generated with an efficiency of 100%and these rescue constructs were targeted via RMCE, to P120 catenin knock out ES cells, with an efficiency of 97%Cystic EB morphology can be used as a phenotypic readout to screen different rescue constructs.As proof of concept, R26-driven P120 catenin isoform 1A rescued the p120 catenin knock out phenotype. To test whether E-cadherin independent membrane anchoring of P120 catenin enabled csytic EB formation, the k-ras membrane targeting motif, CAAX, was fused to the carboxy terminus of P120 catenin, and introduced via RMCE, to P120 catenin knock out ES cells, before EBs were made from them. However, this construct serves a dominant negative that does not allow binding and stabilization of E-cadherin.And as a consequence, does not rescue the p120 catenin knock out phenotype. Epithelial-mesenchymal transition, or EMT, is an essential developmental process that also occurs during ES cell differentiation. When expressed in p120 catenin knock out ES cells, the EMT inducers, ZEB1, ZEB2, or snail that can directly repress various epithelial marker genes like E-cadherin, failed to restore cystic EB formation.Once mastered, RMCE compatible ES cells can be isolated within one month. RMCE compatible targeting vectors can be generated within one week, and targeted rescue of ES cells can be achieved within one month using this technique, if it is performed properly. After watching this video, you should have a good understanding of how to use RMCE to perform structure function studies in naive or differentiated embryonic stem cells.

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