우리는 투과 전자 현미경의 도움 스테판 켄스 감사합니다. 이 작품은 건강 그랜트 R01AG023731의 국립 연구소에 의해 지원되었다.

이전 연구는 Mgm101의 첫 번째 아미노 말단 22 잔기가 미토콘드리아 19로 가져온 후 쪼개 것으로 나타났습니다. 대장균에서 발현를 들어, 첫 번째 22 코돈이 부족한 MGM101 오픈 읽기 프레임은 PCR에 의해 증폭되어 발현 벡터 PMAL - C2E의 수정 된 버전에 말토오스 결합 단백질 (MBP)를 코딩하는 남성 서열의 하류에 배치됩니다. 이 PreScission 프로테아제 (그림 3)를위한 절단 사이트를 포함하는 링커와 MBP-Mgm101 융합을 생성합니다. 플라스미드는 먼저 E.를 선택하여 구성되어 IPTG와 Xgal 화이트 / 블루 선택하지 대장균의 형질 전환. 결과 플라스미드 PMAL - C2E-MGM101 그런 E.에 소개 대장균은 암피실린 및 클로람페니콜 내성 식민지를 선택하여 BL21-CodonPlus (DE3) RIL을 변형. 1. 표현, 유도, 세포 용해와 DNase의 I 트리트먼트 <ol><li> INOC포도당 (0.2 %), 암피실린 (100 ㎍ / ㎖) 및 클로람페니콜 (50 ㎍ / ㎖)를 첨가 LB 배지 10 ㎖ (1 % Bacto 트립 톤, 0.5 % 효모 추출물, 1 %의 NaCl)에 새로운 형질을 ulate. 200 rpm으로 진탕 37 ° CO / N에 품어. </li><li> 위와 같이 보충 LB 배지 2 리터에 10 ㎖의 예비 컬쳐 접종. OD 600이 0.5에 도달 할 때까지 흔들어 37 ° C에서 세포를 성장. </li><li> 0.3 mm의 최종 농도에 이소 프로필 β-D-1-티오 갈 락토 피 라노 시드 (IPTG)를 추가하여 MBP-Mgm101 융합 단백질의 발현을 유도. 5 시간 동안 30 ° C에서 진탕 세포를 성장. </li><li> 베크 JA-10 회 (5,500 XG, 4 ° C, 10 분)를 사용하여 원심 분리하여 세포를 수집합니다. 폐기 후 상층 액은 단백질 분해 효소 억제제 (25 μM의 펩틴, 1 μM의 pepstatin 및 1 ㎜ PMSF)를 포함 용해 버퍼의 30 ML (20 MM 트리스 - 염산, 산도 7.4, 1 mM의 EDTA (에틸렌 다이아 민 테트라 초산), 산도 7.4)에있는 세포 펠렛을 resuspend. </li><li> 20 얼음에 세포 현탁액을 초음파 처리초 초음파 프로세서 (열 시스템, 모델 W-385)를 사용하여 50 % 듀티 사이클에서 30 초 동안 얼음에 냉각하고, 배 반복 할 수 있습니다. </li><li> 2 MG / ML에서 DNase의 1 주식의 1 ML을 추가합니다. 2 시간 동안 4 ° C에서 세포 해물 바위. </li><li> 500 mm의 최종 농도에 NaCl을 조정합니다. </li><li> 30 분 동안 4 ° C에서 10,000 XG에서 원심 분리하여 세포 파편을 제거합니다. </li></ol> 2. 아밀로오스 친 화성 크로마토 그래피로 정제 <ol><li> 용해 버퍼의 아밀로스 수지 (50 % 슬러리)의 1.5 ml의 평형. 세포의 파쇄에 평형 아밀로스 수지를 추가합니다. 4 ° CO / N.에 가볍게 흔들어 </li><li> 찬 방에 설치 이코노 컬럼 크로마토 그래피 칼럼에 해물 수지의 혼합을로드합니다. 언 바운드 단백질이 중력에 의해 열을 전달할 수 있습니다. </li><li> 세척 버퍼 I (; 400 mM의 NaCl을, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산), 산도 7.4, 0.2 mM의 PMSF 20 MM 트리스 - 염산, 산도 7.4) 300 ml의 아밀로오스 수지 씻으십시오. </li><li> 세척 버퍼 II (20 mM의 T 150 ml의 세척을 반복RIS-염산, 산도 7.4, 200 MM의 염화나트륨, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산), 산도 7.4, 0.2 mM의 PMSF). </li><li> 열, 4 품어 10 분 ° C, 수집 용출 버퍼의 5 ML을 (; 200 mM의 NaCl을; 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산), 산도 7.4 및 0.2 mM의 PMSF, 10 MM 토스 20 MM 트리스 - 염산, 산도 7.4)을 추가 더 많은 시간을위한 용출액를 반복하십시오. </li><li> 용출액을 결합, 쿠마시 염색 (그림 4) 다음 SDS-PAGE 젤에 용출액의 나누어지는을로드하여 MBP-Mgm101의 수율과 순도를 결정합니다. </li></ol> 3. PreScission 프로테아제 분열과 양이온 교환 크로마토 그래피 <ol><li> MBP-Mgm101 용출액에 PreScission 프로테아제의 50 단위를 추가합니다. 4의 절단 ° CO / N를 수행하고 (20 MM 트리스 - 염산, 산도 7.4, 100 MM의 염화나트륨, 2 mM의 DTT, 1 mM의 EDTA (에틸렌 다이아 민 테트라 초산), 산도 7.4, 0.2 mM의 PMSF)는 투석 버퍼에 대한 투석 동안 계속할 수 </li><li> SDS-PAGE 젤 (그림 5A)의 분열의 효율성을 확인합니다. </li><li> 여러가 주입에 의해 절단 된 MBP-Mgm101를로드바이오 스케일 미니 매크로 준비 높은 S 카트리지 이온은 바이오 방사선의 생물학적 DuoFlow FPLC 시스템에 연결되어 있습니다. </li><li> 단계 소금 5 mm의 그라데이션, 300 밀리미터, 500 밀리미터, 750 mm와 혼합 버퍼 A (10 mM의 NaCl을하여 만든 염화나트륨 1,000 밀리미터를 적용하여 양이온 교환 크로마토 그래피를 시작, 10 MM 나 - 인산, 산도 7.2, 1 MM의 PMSF) 및 버퍼 B (1 M의 NaCl, 10 MM 나 - 인산, 산도 7.2, 1 ㎜ PMSF). 0.5 ML / 분에서 유량을 설정합니다. Mgm101 분수를 수집하고 SDS-PAGE 젤 (그림 5B)에서 단백질의 순도를 확인합니다. </li></ol> 4. 크기 배제 크로마토 그래피 <ol><li> 양이온 교환 크로마토 그래피에서 단백질 분수를 결합합니다. (; 150 mM의 NaCl을, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산), 산도 7.4, 5 MM 2 - 메르 캅토 에탄올, 0.2 mM의 PMSF 20 MM의 MOPS, 산도 7.0) GF 평형 버퍼에있는 단백질을 Dialyze. </li><li> 4 ° C.에서 3,000 XG에서 회전시켜 ~ 1 ML에 볼륨을 줄이기 위해 VIVASPIN 15R의 여과 스핀 열이 단백질을 집중 </li><li> Mgm101 (을)를로드NA는 크로마토 그래피 버퍼 (; 150 mM의 NaCl을, 5 MM의 β-메르 캅 토 에탄올, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산), 산도 7.4, 0.2 mM의 PMSF 20 MM의 MOPS, 산도 7.0)로 평형화 Superose 6 준비 급료 열을 보정. </li><li> 0.5 ML / 분의 유속으로 실행 크로마토 그래피 버퍼 크기 배제를 시작합니다. </li><li> 정제 Mgm101 피크의 분수를 수집합니다. 단백질의 최종 품질 검사를위한 12 % SDS-PAGE에 분수 분주을로드합니다. </li><li> VIVASPIN 6 단백질을 농축 한 후 빠르게 -80에서 액체 질소와 저장소에 샘플을 동결 ° C. </li><li> ssDNA 결합 분석 (그림 8)의 경우와 투과 전자 현미경 (그림 9)에 의해 구조 시각화를위한 6-12 개월 이내에 Mgm101 샘플을 사용합니다. </li></ol>

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저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

그것은 E.에서 안정적인 기본 재조합 Mgm101 단백질을 생산하기 위해 도전하고있다 박테리아 세포에서의 용해성을 가능성으로 인해 대장균. 이 보고서에서, 우리는 MBP 융합 전략은 단백질이 비교적 높은 수준에서 표현 될 수 있다는 점을 보여줍니다. 부정적인 염색 투과 전자 현미경 및 크기 배제 크로마토 그래피를 사용하여, 우리는 이전에 MBP-융합 단백질은 생체 18 균...

상동 재조합 이중 가닥 나누기의 수리 (DSBs) 및 interstrand 가교를 위해 및 축소 복제 포크 1 DNA 복제의 reinitiation 중요합니다. 기존의 상동 재조합에, 중앙 반응은 원핵 생물에서 RECA 및 Rad51와 1-3 진핵 생물 Dmc1 등 ATP-의존 recombinases에 의해 촉매된다. 이 recombinases 양면 DNA 템플릿 (그림 1 왼쪽 패널) 4-7에서 동성 검색 및 가닥의 침공을 시작하기에 필수적인 ssDNA에 핵 단백질 필라멘트를 형성한다. 기존의 방식에 더하여, 상동 재조합은 RecA/Rad51-independent 방식 (그림 1, 오른쪽 패널)에 자리를 취할 수 있습니다. 예를 들어, 효모 Rad52 및 Rad59 단백질은 직접 dsDNA 나누기 resectioning에 의해 노출되는 보완 ssDNA 가닥의 어닐링을 촉진 할 수 있습니다. 노래로 알려진이 재조합 과정르 가닥 어닐링, 일반적으로 동종 dsDNA 템플릿을 페어링 포함되지 않습니다. 어닐링 후, 이종 꼬리 exonucleases에 의해 제거하고 흠이 게놈 연속성 8-10을 복원 할 결찰하고 있습니다. 한 가닥 어닐링 기계의 수리는 종종 직접적으로 반복 영역 사이의 게놈 시퀀스의 삭제가 붙어 있습니다. Rad52는 살균 11 사이에 널리 퍼져 재조합 단백질의 다양한 그룹에 속한다. 이 단백질은 또한 동종 단일 가닥 DNA 분자의 풀림을 촉진 자신의 활동을 기반으로, 단일 가닥 어닐링 단백질 (SSAPs)로 알려져 있습니다. 가장 특징 박테리오파지 SSAPs는 Redβ 및 lactococcal 파지 ul36에서 prophage RAC와 Sak의 단백질에서 박테리오파지 λ와 P22, 사각형에서 ERF 수 있습니다. 유사성이 거의 undetect 있지만 SSAPs는 구조적으로, 전형적인 β-β-β-α 배로 특징자신의 기본 시퀀스 수. 10 그들은 모두 형태의 대형 호모 중합체 링 - 체외 12-14 14 배 대칭. 이러한 특성 고차 구조 조직의 기능 의미가 잘 이해되지 않습니다. 우리는 미토콘드리아 게놈의 상동 재조합의 메커니즘을 이해에 관심이 있습니다. 우리는 이전에 사카 cerevisiae의 15 미토콘드리아 DNA의 유지에 필수적입니다 MGM101 유전자를 발견했습니다. MGM101은 이후 미토콘드리아 nucleoids과 연관된 것으로 발견되었으며, DNA를 손상 요원 미토콘드리아의 허용 오차 16이 필요합니다. 그러나 Mgm101의 연구는 재조합 Mgm101을 생산하기 위해 어려움 지난 10 년 다시 개최되었습니다. 우리는 최근에 E.에서 대량으로 용해 Mgm101 생산에 성공했다 MBP 융합 전략을 사용 대장균. 이것은 우리가 그 Mgm101 주식을 보여줄 수있게되었습니다단백질 17,18의 Rad52 - 가족과 함께 생화학 및 구조 유사성. 이 보고서에서는 세 단계 정화 절차는 균일 Mgm101for 생화학 및 구조 분석 (그림 2)을 생산하는 기술입니다.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Expression vector pMAL-c2E</td>
			<td>New England Biolabs</td>
			<td>#N8066S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PreScission Protease</td>
			<td>GE Healthcare Life Sciences</td>
			<td>#27-0843-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BL21-CodonPlus(DE3)-RIL cells</td>
			<td>Strategene</td>
			<td>#230245</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Roche Applied Science</td>
			<td>#11034626001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pepstatin</td>
			<td>Roche Applied Science</td>
			<td>#11359053001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Roche Applied Science</td>
			<td>#10837091001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Sigma</td>
			<td>#DN25-1G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropyl &beta;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Roche Applied Science</td>
			<td>#11411446001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amylose resin</td>
			<td>New England Biolabs</td>
			<td>#E8021L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Econo-Column chromatography column</td>
			<td>BIO-RAD</td>
			<td>#7372512</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bio-Scale Mini Macro-Prep High S cartridge (1 ml)</td>
			<td>BIO-RAD</td>
			<td>#732-4130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VIVASPIN 15R Ultrafiltration spin column (10,000 MWCO)</td>
			<td>Sartorius Stedium</td>
			<td>#VS15RH02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superose 6 prep grade column</td>
			<td>Amersham Bioscirnces</td>
			<td>#17-0489-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VIVASPIN 6 Ultrafiltration spin column (5,000 MWCO)</td>
			<td>Sartorius Stedium</td>
			<td>#VS0611</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

preparation of the mgm101 recombination protein by mbp-based tagging strategy

MGM101 유전자는 미토콘드리아 DNA의 유지에있는 그것의 역할을 위해 20 년 전에 발견되었다. 여러 그룹에서 연구 Mgm101 단백질이 미토콘드리아 DNA의 재조합 복구에 관여하는 것을 제안했습니다. 최근 조사 Mgm101이 Rad52 형 재조합 단백질의 가족과 관련된 지적했다. 이 단백질은 큰 올리고머 고리를 형성하고 일치하는 단일 가닥 DNA 분자의 풀림을 촉진합니다. 그러나 Mgm101의 특성은 재조합 단백질을 생산의 어려움에 의해 방해되었다. 여기 재조합 Mgm101의 준비를위한 믿을 수있는 절차를 설명합니다. 말토오스 결합 단백질은 (MBP) 태그 Mgm101 먼저 대장균에서 발현된다. 융합 단백질은 처음 아밀로오스 친 화성 크로마토 그래피로 정제한다. 단백질 가수 분해 분열에 의해 발표 된 후, Mgm101는 양이온 교환 크로마토 그래피로 MBP에서 분리됩니다. 단 분산 Mgm101은 다음 얻을 수있다크기 배제 크로마토 그래피 있습니다. 세균성 문화의 리터 당 Mgm101의 ~ 0.87 밀리그램의 수익률은 정기적으로 얻을 수 있습니다. 재조합 Mgm101는 DNA 최소한의 오염을 보유하고 있습니다. 준비된 샘플이 성공적으로 Mgm101의 생화학 적 구조 및 단일 입자 이미지 분석에 사용됩니다. 이 프로토콜은 또한으로 misfolded 및 박테리아 세포에 독성이 될 수있는 다른 큰 올리고머 DNA-결합 단백질의 준비를 위해 사용될 수있다.

Mgm101은 미토콘드리아에서 Rad52 관련 재조합 단백질이다. Rad52은 광범위 미토콘드리아 DNA 재조합 (그림 1)에서의 역할에 대해 연구하고있다. 재조합 Mgm101은 세 단계의 절차 (그림 2)에 의해 제조 될 수있다. 이 Mgm101는 수용성 형태로 표현하고 이후 (그림 3) 단백질 가수 분해 분열에 의해 태그에서 해제 할 수 있습니다 MBP를 붙이는 전략의 사용에 의해 촉진된다. <...

Watch this Scientific Journal Video about Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy at JoVE.com

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

효모 미토콘드리아 핵 양체 단백질, Mgm101이 큰 올리고머 고리를 형성하는 Rad52 형 재조합 단백질이다. 프로토콜은 말토오스 결합 단백질 (MBP) 태그 양이온 교환 및 크기 배제 크로마토 그래피와 결합 전략을 사용하고 수용성 재조합 Mgm101 준비를 설명합니다.

MBP 기반 태깅 전략에 의해 Mgm101 재조합 단백질의 제조

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

preparation-mgm101-recombination-protein-mbp-based-tagging

Research

JoVE Journal

Biology

12.0K 조회수.  State University of New York Upstate Medical University. 효모 미토콘드리아 핵 양체 단백질, Mgm101이 큰 올리고머 고리를 형성하는 Rad52 형 재조합 단백질이다. 프로토콜은 말토오스 결합 단백질 (MBP) 태그 양이온 교환 및 크기 배제 크로마토 그래피와 결합 전략을 사용하고 수용성 재조합 Mgm101 준비를 설명합니다.

비디오: Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

연구

생물학

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

내생 와일드 타입 BRCA1을 표현 세포를 사용하는 동종 재조합에 BRCA1 변이의 효과를 확인

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

침샘 조직 문화의 유전자 변형과 재결합

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

에서 상동 재조합 구문을 Recombineering<em&gt; 초파리</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

임의 Microseed 매트릭스 심사에 의해 단백질 결정화의 성공률을 향상

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4&#039;,6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

녹색 형광 단백질 태그와 4', 6-Diamidino-2-phenylindole 꼬마 선 충 에 얼룩이 단백질 Subcellular 지 방화를 감지

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

유세포분석으로 생체내 단백질 자가 조립 검출 및 특성화

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

루트 엑수데이트에서 뿌리 균 화학 물질의 신속 한 식별에 대 한 향상 된 화학 분석

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...