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><tbody><tr><td>Expression vector pMAL-c2E</td><td>New England Biolabs</td><td>#N8066S</td><td></td></tr><tr><td>PreScission Protease</td><td>GE Healthcare Life Sciences</td><td>#27-0843-01</td><td></td></tr><tr><td>BL21-CodonPlus(DE3)-RIL cells</td><td>Strategene</td><td>#230245</td><td></td></tr><tr><td>Leupeptin</td><td>Roche Applied Science</td><td>#11034626001</td><td></td></tr><tr><td>Pepstatin</td><td>Roche Applied Science</td><td>#11359053001</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Roche Applied Science</td><td>#10837091001</td><td></td></tr><tr><td>DNAse I</td><td>Sigma</td><td>#DN25-1G</td><td></td></tr><tr><td>Isopropyl &amp;#946;-D-1-thiogalactopyranoside (IPTG)</td><td>Roche Applied Science</td><td>#11411446001</td><td></td></tr><tr><td>Amylose resin</td><td>New England Biolabs</td><td>#E8021L</td><td></td></tr><tr><td>Econo-Column chromatography column</td><td>BIO-RAD</td><td>#7372512</td><td></td></tr><tr><td>Bio-Scale Mini Macro-Prep High S cartridge (1 ml)</td><td>BIO-RAD</td><td>#732-4130</td><td></td></tr><tr><td>VIVASPIN 15R Ultrafiltration spin column (10,000 MWCO)</td><td>Sartorius Stedium</td><td>#VS15RH02</td><td></td></tr><tr><td>Superose 6 prep grade column</td><td>Amersham Bioscirnces</td><td>#17-0489-01</td><td></td></tr><tr><td>VIVASPIN 6 Ultrafiltration spin column (5,000 MWCO)</td><td>Sartorius Stedium</td><td>#VS0611</td><td></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

Universidad de Cartagena UDEC

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

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

인공 요소 엔지니어 것은 특히 인간 세포에서의 스 플라이 싱을 조작 할

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

연구

유전학

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

유도 성 T7 RNA 중합 효소 매개 멀티젠 발현 시스템, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9-중재 대상된 통합에서 Vivo에서 Homology 중재 끝에 합류 기반 전략을 사용 하 여

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

유전 조작<em&gt; Δku80</em의 기능 게놈 분석을위한&gt; 균주<em&gt; 톡소 포자충</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

면역학 및 감염병학

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

신경 모세포종 전이의 제브라피시 모델

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

암 연구학

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

이분자 형광 보완과 광 활성화 현지화 현미경 (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

생체공학

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.

This protocol details the enrichment of native mycobacterial extracellular vesicles (mEVs) from axenic cultures of Mycobacterium smegmatis (Msm) and how mCherry (a red fluorescent reporter)-containing recombinant MsmEVs can be designed and enriched. Lastly, it verifies the novel approach with the enrichment of MsmEVs containing the EsxA protein of Mycobacterium tuberculosis.

마이코박테리아의 native and recombinant extracellular vesicles 농축

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

서브 클로닝 플러스 삽입 (SPI) - 유전자 타겟팅 벡터의 신속한 건설 소설 Recombineering 방법

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

생물학

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that allow the tagging of endogenous genes to produce fluorescent fusion proteins. These methods enable researchers to visualize protein behavior in real time, providing valuable insights into their functions and interactions within the cellular environment. Many current gene tagging studies employ a two-step process where visible markers, such as eye color changes, are used to identify genetically modified organisms in the first step, and the visible marker is excised in the second step. Here, we present a one-step protocol to perform precise and rapid endogenous gene tagging in Drosophila melanogaster, which enables screening for engineered lines without the visible eye marker, offering a significant advantage over past methods. To screen for successful gene-tagging events, we employ a PCR-based technique to genotype individual flies by analyzing a small segment from their middle leg. Flies that pass the screening criteria are then used to produce stable stocks. Here, we detail the design and construction of CRISPR editing plasmids and methods for screening and confirmation of engineered lines. Together, this protocol improves the efficiency of endogenous gene tagging in Drosophila significantly and enables studies of cellular processes in vivo.

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Here, we present a simplified endogenous gene tagging protocol for Drosophila, which utilizes a PCR-based technique for marker-free identification of successful genetic modifications, facilitating the development of stable knock-in lines.

Drosophila melanogaster의 내인성 유전자 태깅을 위한 원스텝 CRISPR 기반 전략

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that ...

신경과학

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

MBP 기반 태깅 전략에 의해 Mgm101 재조합 단백질의 제조

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