Name: protocol for microrna transfer into adult bone marrow-derived hematopoietic stem cells to enable cell engineering combined with magnetic targeting
Uploaded: 2018-06-18T13:00:00.000+00:00
Duration: 11 min 37 s
Description: Watch this Scientific Journal Video about Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting at JoVE.com

이 작품은 연방 교육부 및 연구 (FKZ 0312138A 및 FKZ 316159), EU 구조 기금 (ESF/IVWM-B34-0030/10와 ESF/IVBM-B35-0010/12), 주 Mecklenburg 서쪽 Pomerania 독일과 DFG (DA1296/2-1)에 의해 지원 되었다는 독일 심장 재단 (F/01/12), BMBF (VIP + 00240) 고 젖은 기초. 또한, F.H. 및 오후는 FORUN 프로그램의로 스톡 대학 의료 센터 (889001)에 의해 지원 됩니다.

저자는 충돌의 관심을 선언합니다.

최근 몇 년 동안, CD133+ SCs 등장 유망한 세포 인구로 SC 기반 요법으로 입증 단계 I, II, 및 III에 대 한 임상 시험43,44,,4546, 47 , 48 , 49 , 50 , 51 , ...

CD133+ 이종 줄기 재생 의학에 대 한 잠재력 약속 된 조상 세포 인구를 대표 하는 SCs. 그들의 조 혈, 내 피, 및 조직적 분화 잠재적인1,2,3 수는 CD133+ 세포, 예를 들어, 차별화를 통해 neovascularization 프로세스에 기여 하 으로 새로 형성 선박 및 프로-신생 paracrine 메커니즘4,5,,67에 의해 신호의 활성화.
30 개 이상의 승인 된 임상 시험 (ClinicalTrails.gov)에서 그들의 높은 잠재력에도 불구 하 고 그들의 치료 결과 중인 여전히 논쟁 적인 토론4. 실제로, SCs의 임상 응용 프로그램 낮은 보존 관심과 대규모 초기 세포 죽음5,,89의 기관에 의해 방해 된다. 추가 CD133의 엔지니어링+ SCs 사전 이식 이러한 난제를 극복 도움이 될.
효율적인 세포 치료에 대 한 전제 조건 치료 관련 셀10engraftment를 향상 시키기 위해 대규모 초기 세포 죽음의 감소 될 것 이다. 현재 연구 시연 첫 1-2 h, 이식된 세포 유형의 독립적인 동안 매우 끼얹는다 뇌와 심장 등 장기에 90-99%의 엄청난 셀 손실 또는 응용 프로그램 경로11,12,13 14,15,16,17,18,19,,2021. 관심22,23,,2425,26의 사이트에 대상 셀에 혁신적인 비-침략 적 전략을 가능 하 게 SC 마그네틱 나노 (MNPs)를 사용 하 여 라벨 고 동시에 셀 MRI27 (MPI) 이미징 자성 입자를 사용 하 여 모니터링 하는 것을 허용 한다. 가장 효율적인 vivo에서 정 맥 주입23,,2428 후 셀 지도 보다 로컬 관리 후 사용한 셀 보존 대상 적용 자성된 셀 연구 . 따라서, 우리의 그룹 superparamagnetic 산화 철 나노 입자29의 구성 된 배달 시스템을 설계. 이 기술로, CD133+ SCs와 인간 탯 줄 정 맥 내 피 세포 (HUVECs)는 대상 효율적으로 수, 여 체 외에 시도30,31.
사우스 캐롤라이나 요법에 대 한 또 다른 장애물 초기 세포 죽음32에 기여 하는 이식 후 영향을 받는 조직의 적대적인 염증 성 환경입니다. 여러 사전 컨디셔닝 연구 뿐만 아니라 치료 관련 미르의 응용 프로그램은 테스트33; 그것은 성공적으로 입증 되었습니다 안티 apoptotic 미르 생체 외에서 apoptosis를 억제 고 vivo에서세포 engraftment33을 강화. 이 작은 분자, 20-25의 뉴클레오티드로 구성 메신저 RNAs (mRNAs)의 posttranscriptional 변조기로 서 중요 한 역할을 하 고 따라서 줄기 세포 운명과 행동34를 영향을. 또한, 미르 세 도입 호스트 게놈34에 원치 않는 안정적인 통합을 하지 마십시오.
기본 SCs에 핵 산 (NAs)의 효율적인 도입에 대 한 현재 시도 주로 재조합 바이러스8,35기반으로 합니다. 높은 transfection 효율에도 불구 하 고 재조합 바이러스 조작은 벤치를 머리 맡 번역, 예를 들면, 통제 유전자 발현, pathogenicity, immunogenicity, 및 insertional mutagenesis35에 대 한 주요 장애물이 선물 ,36. 따라서, 폴리머 기반 구조 등 비 바이러스 성 전달 시스템 개발에 중요 하다. 그 중에서 polyethylenimine (페이) 저하, 세포질 통풍 관에서에서 보호 하기 위해 나 결로 등 미르에 대 한 혜택을 제공 하는 유효한 배달 차량 나타내고 endosomal 통해 세포내 릴리스 탈출37,38. 또한, 미르-페이 단지 임상 시험39높은 생체 적합성을 설명 했다. 따라서, 우리의 배달 시스템은 biotinylated의 구성 분기 25 kDa 페이는 streptavidin 입히는 MNP 코어30,,3140에 바인딩된.
이 원고에서는 CD133의 (i)는 수동 격리를 설명 하는 포괄적인 프로토콜 소개+ 사우스 캐롤라이나 사우스 캐롤라이나 제품과의 효율적이 고 부드러운 transfection 전략 (ii)의 상세한 묘사와 인간의 골 수 (BM) 기부에서 한 자석으로 비 바이러스 성 고분자 기반 배달 시스템 CD133의 유전 공학에 대 한+ SCs 미르를 사용 하 여. CD133+ SCs 격리 되며 표면 항 체 기반 자기 활성화 셀 정렬 (맥) 시스템을 사용 하 여 인간의 sternal BM aspirates에서 자석으로 농축. 이후에, 세포 생존 능력 뿐만 아니라 셀 순도 cytometry 사용 하 여 분석 된다. 그 후, 미르/PEI/MNP 단지 준비가 CD133+ SCs는 페. transfection 후 18 h, 통풍 관 효율성과 transfection SC 마커 표현과 세포 생존 능력에의 영향 분석 된다. 또한, transfection 복잡 한 화합물의 세포내 분포의 평가 4 색 레이블 및 구조화 조명 현미경 (SIM)를 사용 하 여 수행 됩니다.

<table><tbody><tr><td>7-AAD</td><td>BD Biosciences</td><td>559925</td><td></td></tr><tr><td>Acetic Acid with Methylene Blue</td><td>Stemcell Technologies</td><td>7060</td><td>3%</td></tr><tr><td>anti-CD133/2-PE (clone: 293C3)</td><td>Miltenyi Biotec GmbH</td><td>130-090-853</td><td></td></tr><tr><td>anti-CD34-FITC (clone: AC136)</td><td>Miltenyi Biotec GmbH</td><td>130-081-001</td><td></td></tr><tr><td>anti-CD45-APC-H7 (clone: 2D1)</td><td>BD Biosciences</td><td>560178</td><td></td></tr><tr><td>rhodamine dye; Atto 565 dye conjugated to biotin</td><td>ATTO-TEC GmbH</td><td>AD 565-71</td><td></td></tr><tr><td>BD FACS LSRII flow cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BD FACSDiva Software 6.1.2</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich GmbH</td><td>A7906</td><td></td></tr><tr><td>CD133 antibody-linked superparamagnetic iron dextran particles; CD133 MicroBead Kit</td><td>Miltenyi Biotec GmbH</td><td>130-097-049</td><td></td></tr><tr><td>collagenase B</td><td>Roche Diagnostics GmbH</td><td>11088831001</td><td></td></tr><tr><td>counting chamber</td><td>Paul Marienfeld GmbH &amp;amp; Co. KG</td><td></td><td></td></tr><tr><td>Cyanine 3 dye labelled precursor miR; Cy3 Dye-Labeled Pre-miR Negative Control #1</td><td>Ambion</td><td>AM17120</td><td></td></tr><tr><td>Cyanine 5 dye miR labelling kit; Cy5 dye Label IT miRNA Labeling Kit</td><td>Mirus Bio</td><td>MIR 9650</td><td></td></tr><tr><td>DNAse I</td><td>Roche Diagnostics GmbH</td><td>10104159001</td><td>(100 U/mL)</td></tr><tr><td>ELYRA PS.1 LSM 780 confocal microscope</td><td>Carl Zeiss Jena GmbH</td><td></td><td></td></tr><tr><td>FcR Blocking Reagent, human</td><td>Miltenyi Biotec GmbH</td><td>130-059-901</td><td></td></tr><tr><td>bright green protein labeling kit; Oregon Green 488 Protein Labeling Kit</td><td>Thermo Fisher Scientific</td><td>O10241</td><td></td></tr><tr><td>aqueous mounting medium; Fluoroshield</td><td>Sigma-Aldrich GmbH</td><td>F6182</td><td></td></tr><tr><td>density gradient centrifugation tube; Leukosep Centrifuge Tube</td><td>Greiner Bio-One</td><td>89048-932</td><td></td></tr><tr><td>MACS magnet holder; MACS MultiStand</td><td>Miltenyi Biotec GmbH</td><td>130-042-303</td><td></td></tr><tr><td>MACS pre-separation filter</td><td>Miltenyi Biotec GmbH</td><td>130-041-407</td><td>30 &amp;micro;m</td></tr><tr><td>MACS separation column (MS / LS)</td><td>Miltenyi Biotec GmbH</td><td>130-042-201 / 130-042-401</td><td></td></tr><tr><td>MACS permanent magnet; MACS Separator</td><td>Miltenyi Biotec GmbH</td><td>130-042-302</td><td></td></tr><tr><td>Millex-HV PVDF Filter</td><td>Merck</td><td>SLHV013SL</td><td>0.45 &amp;mu;m</td></tr><tr><td>mouse IgG 2b-PE</td><td>Miltenyi Biotec GmbH</td><td>130-092-215</td><td></td></tr><tr><td>amine reactive dye; Near-IR LIVE/DEAD Fixable Dead Cell Stain Kit</td><td>Thermo Fisher Scientific</td><td>L10119</td><td></td></tr><tr><td>human lymphocyte separating medium; Pancoll</td><td>Pan Biotech GmbH</td><td>P04-60500</td><td>density: 1.077 g/mL</td></tr><tr><td>PBS</td><td>Pan Biotech GmbH</td><td>P04-53500</td><td>without Ca and Mg</td></tr><tr><td>PEI</td><td>Sigma-Aldrich GmbH</td><td>408727</td><td>branched; 25 kDa</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>100 U/mL, 100 &amp;mu;g/mL</td></tr><tr><td>PFA</td><td>Merck Schuchardt OHG</td><td>1040051000</td><td></td></tr><tr><td>unlabelled precursor miR; Pre-miR miRNA Precursor Negative Control #1</td><td>Ambion</td><td>AM17110</td><td></td></tr><tr><td>RBC lysis buffer</td><td>eBioscience</td><td>00-4333-57</td><td></td></tr><tr><td>RNAse decontamination solution; RNaseZap</td><td>Thermo Fisher Scientific</td><td>AM9780</td><td></td></tr><tr><td>human lymphocyte medium; Roswell Park Memorial Institute (RPMI) 1640 medium</td><td>Pan Biotech GmbH</td><td>P04-16500</td><td></td></tr><tr><td>recombinant human cytokine supplement; StemSpan CC100</td><td>Stemcell Technologies</td><td>2690</td><td></td></tr><tr><td>serum-free haematopoietic cell expansion medium; StemSpan H3000</td><td>Stemcell Technologies</td><td>9800</td><td></td></tr><tr><td>Streptavidin MagneSphere Paramagnetic Particles</td><td>Promega Corporation</td><td>Z5481</td><td></td></tr><tr><td>Trypan Blue solution</td><td>Sigma-Aldrich GmbH</td><td>T8154</td><td>0.4 %</td></tr><tr><td>UltraPure EDTA</td><td>Thermo Fisher Scientific</td><td>15575020</td><td>0.5 M; pH 8.0</td></tr><tr><td>ZEN2011 software</td><td>Carl Zeiss Jena GmbH</td><td></td><td></td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Sonorex RK 100 SH sonicating water bath</td><td>Bandelin electronic</td><td></td><td>Ultrasonic nominal output: 80 W; Ultrasonic frequency: 35 kHz</td></tr></tbody></table>

protocol for microrna transfer into adult bone marrow-derived hematopoietic stem cells to enable cell engineering combined with magnetic targeting

동안 CD133+ 조 혈 줄기 세포 (SCs) 재생 의학 분야에서 높은 잠재력을 제공 하기 위해 입증 되었습니다, 그들의 낮은 유지 비율 관찰된 대규모 세포 사망률 부상된 조직에 주입 후 매우 제한 된 치료 효과입니다. 이러한 한계를 극복 하기 위해 우리가 그들의 관리에 앞서 적당 한 셀 엔지니어링에 대 한 비 바이러스 성 기반된 프로토콜을 확립 하고자 했다. 인간의 CD133 수정+ 셀의 대상 잠재력 뿐만 아니라 통풍 관 효율 및 안전에 관하여 해결 SCs 예측에 관한 (미르) 로드 자석 polyplexes를 사용 하 여 표현. 우리의 프로토콜에 의존, 우리는 CD133 동안 80-90%의 높은 미르 통풍 관 비율을 달성할 수 있는+ 줄기 세포 속성을 받지 않습니다. 또한,이 수정 된 세포는 자기 대상의 옵션을 제공합니다. 우리 여기 CD133의 수정에 대 한 안전 하 고 매우 효율적인 절차 설명+ SCs. 우리는 줄기 세포 치료 효과의 최적화에 대 한 자기 공명 영상 (MRI)을 통해 관리 셀 제품의 모니터링을 위한 표준 기술 제공이 접근을 기대 합니다.

수동 격리와 인간의 BM 파생 CD133의 자석 농축 제시 프로토콜 설명+ SCs 후속 바이러스 독립 셀에 체 외에서 세포 조작 및 에 대 한 비-침략 적 기술 전략, 엔지니어링 vivo 모니터링 도구.
이 3 단계 절연 기술 조밀도 기온 변화도 원심 분리를 통해 미리 소화 sternal BM에서 MNCs의 분리를 허용합니다. 이후에, CD133<...

Watch this Scientific Journal Video about Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting at JoVE.com

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

이 프로토콜에서는 안전 하 고 효율적인 절차 CD133 수정+ 조 혈 줄기 세포. 제시 비 바이러스 성, 자기 polyplex 기반 접근 자기 공명 화상 진 찰을 통해 관리 셀 제품 모니터링에 관해서는 뿐만 아니라 치료 줄기 세포 효과의 최적화를 위한 기반을 제공할 수 있습니다.

성인 골 수 유래 조 혈 줄기 세포로 세포 공학 자기 대상으로 함께 있도록 예측에 관한 전송을 위한 프로토콜

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

protocol-for-microrna-transfer-into-adult-bone-marrow-derived

Research

JoVE Journal

Bioengineering

6.6K 조회수.  Rostock University Medical Center. 이 프로토콜에서는 안전 하 고 효율적인 절차 CD133 수정+ 조 혈 줄기 세포. 제시 비 바이러스 성, 자기 polyplex 기반 접근 자기 공명 화상 진 찰을 통해 관리 셀 제품 모니터링에 관해서는 뿐만 아니라 치료 줄기 세포 효과의 최적화를 위한 기반을 제공할 수 있습니다.

비디오: Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

전사 인자와 마우스 배아 섬유 아 세포를 재 프로그램하는 것은 Hemogenic 프로그램을 유도하는

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

연구

발생학

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

절연, 특성화 및 예측에 관한 기반 유전자 조작 인간 치과 여 포 줄기 세포의

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

유전학

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

격리, 시퀀싱 및 인간 중간 엽 줄기 세포에서 세포 외 MicroRNAs 분석

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

준비 및 Nonhuman 영장류 조 혈 줄기와 조상 세포의 유전자 수정

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

A Guide to Precise Gene Disruption in Primary Bone Marrow-Derived macrophages(원발성 골수 유래 대식세포의 정확한 유전자 파괴에 대한 가이드)

High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes

Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the physiology of macrophages in vivo more closely than immortalized macrophage cell lines and can be derived from mice already carrying defined genetic changes. However, disrupting gene function in BMDMs remains technically challenging. Here, we provide a protocol for efficient CRISPR/Cas9 genome editing in BMDMs, which allows for the introduction of small insertions and deletions (indels) that result in frameshift mutations that disrupt gene function. The protocol describes how to synthesize single-guide RNAs (sgRNA-Cas9) and form purified sgRNA-Cas9 ribonucleoprotein complexes (RNPs) that can be delivered by electroporation. It also provides an efficient method for monitoring editing efficiency using routine Sanger sequencing and a freely available online analysis program. The protocol can be performed within 1 week and does not require plasmid construction; it typically results in 85% to 95% editing efficiency.

저자 스포트라이트: 정확한 유전자 파괴를 위한 골수 유래 대식세포의 효율적인 CRISPR/Cas9 게놈 편집

This protocol describes the procedure for genome editing in mouse bone marrow-derived macrophages using Cas9-sgRNA ribonucleoprotein complexes assembled in vitro and delivered by electroporation.

전기천공된 Cas9-sgRNA 복합체를 사용한 일차 골수 유래 대식세포의 고효율 유전자 파괴

Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

셀 라벨 및 초상 자성 산화철 나노 입자로 타겟팅

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

생체공학

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

인간 조혈 줄기 및 전구 세포에서 발암성 이형접합체 기능 획득 돌연변이 공학

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

암 연구학

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

성인 골 수 유래 조 혈 줄기 세포로 세포 공학 자기 대상으로 함께 있도록 예측에 관한 전송을 위한 프로토콜

요약

더 많은 비디오 탐색

이 비디오의 챕터

전사 인자와 마우스 배아 섬유 아 세포를 재 프로그램하는 것은 Hemogenic 프로그램을 유도하는

절연, 특성화 및 예측에 관한 기반 유전자 조작 인간 치과 여 포 줄기 세포의

격리, 시퀀싱 및 인간 중간 엽 줄기 세포에서 세포 외 MicroRNAs 분석

준비 및 Nonhuman 영장류 조 혈 줄기와 조상 세포의 유전자 수정

전기천공된 Cas9-sgRNA 복합체를 사용한 일차 골수 유래 대식세포의 고효율 유전자 파괴

전기천공된 Cas9-sgRNA 복합체를 사용한 일차 골수 유래 대식세포의 고효율 유전자 파괴

전기천공된 Cas9-sgRNA 복합체를 사용한 일차 골수 유래 대식세포의 고효율 유전자 파괴

셀 라벨 및 초상 자성 산화철 나노 입자로 타겟팅

인간 조혈 줄기 및 전구 세포에서 발암성 이형접합체 기능 획득 돌연변이 공학

식별 및 Oligopotent 및 계보 커밋된 골수성 창시자 마우스 골 수에서 격리