샌들러 천식 기초 연구 센터 (세이버) 샌들러 재단

1 부 : Lentiviruses 생산 HEK 293 세포의 Transfection * 24시간 50~70% 다음날의 confluency에 10cm 접시에 transfection, 플레이트 2.5 X 10 6 세포 전에. <ol><li> 하나는 나중에 바이러스를 하나의 세포를 transfect 것입있는 DNA를 준비하여이 절차를 시작합니다. 첫째, FuGENE 6 Transfection 시약 로체, 세포가 DNA를 잡으 지질 시약을 희석. 1.5 ML 튜브에 피펫 30μl FuGENE 6 600ul 혈청없는 매체로 (SFDMEM). 그것이 매체에 전달로 FuGENE은 튜브의 측면을 건드리지 못하게하는주의하십시오. 이 FuGENE 5 분 실온에서 SFDMEM 믹스와 부화하자. </li><li> 튜브의 뚜껑에서 4 μg 각 제 3 세대 바이러스성 포장 벡터 (pMDL, pRSV 및 pVSV - G)와 플라스미드 lentiviral 4 μg를 섞는다. lentiviral 플라스미드는 DNA 포장 벡터가 lentivirus하기 위해 필요한 다른 모든 단백질에 대한 유전자를 포함하는 동안 바이러스가, 그것이 감염 모든 세포의 게놈에 삽입됩니다 포함되어 있습니다. </li><li> 반전 튜브 몇 배 뚜껑과 믹스를 닫습니다. </li><li> 실온에서 15-20분을 품어. </li><li> 293 판에 튜브의 피펫 전체 내용. 앞뒤로 기울이기 판으로 가볍게 섞는다. </li><li> 인큐베이터에 세포를 반환하고 바이러스 생산 수확하기 전에 48~96시간 위해 계속하실 수 있습니다. </li><li> 부화 기간 동안, 293 세포는 lentiviral 구조의 RNA 사본을 만드는, lentiviral 플라스미드에서 관심의 DNA를 고쳐 쓰다. 그들은 또한 녹음 및 바이러스 단백질로 포장 벡터의 유전자를 번역. 이 단백질은 바이러스가 녹음을 반대하고 감염의 모든 세포의 genomes에 삽입합니다 귀하의 lentiviral 구조의 RNA의 버전을 포함하는 바이러스를 만들기 위해 진행합니다. </li></ol> 2 부 : Lentiviral 하베스트 <ol><li> 잠복기 후, 배양기의 세포를 가지고 이제 바이러스를 포함하는 표면에 뜨는를 제거하는 일회용, 10ml의 주사기를 사용합니다. 뜨는의 약 10ml있을 것입니다. </li><li> 팁에 0.45μM 주사기 필터를 부착하고 베크맨 초원 심 분리기 튜브에 바이러스를 필터링합니다. 필터는 바이러스를 허용하고, 세포는 통과하지 않습니다. </li><li> 폐기하기 전에 모든 바이러스 생산 세포를 품어, 문화 접시, 주사기 45 분 10 % 표백제에 필터. </li></ol> 파트 3 : Ultracentrifugation 통해 Lentiviral 농도 <ol><li> SW - 41Ti 또는 SW - 28 로터 버킷으로 원심 분리기 튜브를로드합니다. </li><li> 모든 바이러스를 포함하는 균형을 0.2g 이내로 튜브를 혈청이없는 DMEM을 사용합니다. </li><li> 초원 심 분리기 내부 회전자를 배치하기 전에 로터 양동이 안으로 들어가서 로터에 그들을 연결 인감 </li><li> 4 초원 심 분리기 90 분 동안 바이러스를 스핀 ° SW - 41Ti 로터에서 25,000 rpm 또는 SW - 28 로터에서 24,000 rpm으로 C. </li><li> 조직 문화 후드에서 10 % 표백제가 들​​어있는 용기에 뜨는 잔을 거꾸로 튜브를 반전. </li><li> 펠렛 주위에 여분의 액체를 흡수하고 5 분 더 이상 편리한 랙에 튜브를 반전하는 Kimwipe을 사용합니다. </li><li> 바이러스 펠렛을 다시 중지하려면 20 시간을 아래 피펫 후, 튜브 100 μL 멸균 PBS를 추가하는 필터 팁을 사용합니다. </li><li> 4 바이러스를 남겨 ° C 다시 중단을 완료 하룻밤. 하나는 약 1 주일 동안 최대 1 년 정도에 대해 -80 4 도에 바이러스 preps를 저장할 수 있습니다. 폐기하기 전에 10 %의 표백제 모든 빈 튜브를 치료해야합니다. </li><li> 한 일주 이상에 대한 바이러스 준비를 사용하고자하는 경우, 적정에 대한 5μl 미래의 실험 3 1 나누어지는 5 - 20μl의 최소 aliquots합니다. 고정 및 마이너스 80 aliquots를 저장합니다. </li></ol> 4 부 : 유동세포계측법 사용 Lentiviral 적정 <ol><li> 다음 단계는 유동세포계측법하여 바이러스 titer를 결정하는 것입니다. 이 방법은 lentiviral 플라스미드는 형광 리포터 유전자를 포함 경우에 작동합니다. 아이디어는 바이러스가 세포를 감염 때, 그 세포의 게놈에 lentiviral 플라스미드 DNA를 도입한다는 것입니다. 감염된 세포는 다음 녹음 및 lentiviral 플라스미드에 포함 유전자를 번역하고 형광 리포터 유전자를 포함하는 경우, 감염된 세포는 형광 것이다. 형광의 수준은 바이러스의 힘, 또는 titer를 나타냅니다. </li><li> 각각의 바이러스 재고가 titered하기 위해서는, 전체 미디어 1.5 ML (DMEM, 펜 - strep, 글루타민, FBS)에 집중 바이러스 3 μl를 희석. </li><li> 96 잘 접시의 단일 행에 우물 1-6에서는 다음과 같은 일련 dilutions을 수행 완료 DMEM 미디어 100 μl를 추가할 모든 6 웰스 수 있습니다. 좋지 100 μl를 추가합니다 물론 두 번째로 (이것은 당신의 흠없는 컨트롤)없이 바이러스와 미디어 100μl를 추가 세번째로 희석 바이러스 100 μl를 (undiluted 바이러스의 상당 to1 ML되는) 추가 먼저물론 두 번째의 혼합의, 잘 잘 다섯 번째로, 잘 3에서 100 ML을 추가할 잘 4 우물에서, 그리고 여섯 번째에 100μl를 추가합니다 물론 5에서 100 μl를 추가 네번째 수 있습니다. 마지막으로 6 자 밖으로 100ul을 가지고 표백제로 폐기. 각 물론 그 전에 한에 비해 바이러스의 절반 볼륨을 가지고 어디에 시리얼 희석이 수행되었다. </li><li> 200μl의 모든 우물에서 전체 볼륨을 가져와. 이들이 가장 집중적으로 바이러스를 titering 위해 잘 작동해야만이 제안 dilutions,됩니다. 바이러스 titer에서 낮은, 또는 unconcentrated 경우 하나는 dilutions을 조정해야 할 수도 있습니다. </li><li> 생물 학적 폐기물 용기에 폐기하기 전에 45 분 동안 10 % 표백제와 함께 사용하지 않은 희석 바이러스를 처리합니다. </li><li> 6 우물 각 ~ 15,000 건강한 적극적으로 나누어 293 세포를 추가합니다. 단일 96 - 웰 플레이트는 16 바이러스 preps을 titer하는 데 사용할 수 있습니다. </li><li> 5 % CO로 37 정도의 배양기에 세포를 반환 2 감염이 최소 48 시간 동안 진행하실 수 있습니다. </li><li> 부화 후, 세포는 흐름 cytometer와 함께 기자의 표현 (우리의 경우 형광의)에 대한 분석입니다. 바이러스성 입자가 잘마다 형광 세포의 퍼센트 quantitated 있습니다. </li></ol> 다음 수식과 ML 당 전염성 단위의 개수를 계산 : <div style="text-align:center;"> (# 감염 X 기자 긍정적인 세포의 날에 잘 당 세포) X 1000 ______________________________________________________ 벡터의 ML이 잘 추가 </div> 제 5 부 : 대표 결과 : 48 시간 잠복기 게시물 transfection 후, 293 세포의 플레이트는 90 % 정도 형광등에 있어야한다. 형광 세포의 비율이 높으면 바이러스 생산 세포의 높은 비율을 나타냅니다. resuspending 때 원심 분리 후, 바이러스 펠렛 겨우 볼 수 있습니다. 펠렛은 투명하고 아주 작은므로 이것은 정상입니다. 세포 세포마다 한 병 이상은 안된다 바이러스 통합을받은 (형광) 감염 때 하나에만 정확한 titer 계산을 달성할 수있는 흐름 cytometer 결과를 해석, 적어 둡니다. 이 경우되도록하기 위해, 계산을위한 &lt;15 % 감염 속도 세포를 사용합니다. 높은 감염률과 세포는 가능성이 세포마다 여러 바이러스 integrants을 받았습니다. 2 단계에서 제안 dilutions는 감염의 범위 내에서 몇 가지 샘플을 얻을 것입니다. 이상적으로, 당신이 마지막 titer를 계산 할 수있는 적정 계획 (선형 라인)을 생성하는 몇 가지 샘플을 사용하지만, 필요하다면 당신은 하나의 샘플을 사용하여 titer를 계산하실 수 있습니다. 하나는 기대할 수 titer 1.0 X 10 7 TU / 취소 집중 바이러스 ML 및 1.0 X 10 8 농축 바이러스 TU / ML.

<ol>
	<li>Reiser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+concentration+of+pseudotyped+HIV-1-based+gene+transfer+vectors..">Production and concentration of pseudotyped HIV-1-based gene transfer vectors..</a> Gene Therapy. , 72-7211 (2000).</li><li>Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D., Naldini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Virol. 72 (11), 8463-8471 (1998).</li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM HEK 293T cells Penicillin-streptomycin Glutamine Phosphate-buffered saline FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FuGENE 6 transfection reagent pSico mCherry pMDL pRSV pVSVG</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bleach</td>
<td>&nbsp;</td>
<td>Clorox</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10-cm cell culture dishes 96 well cell culture dishes </td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353003</td>
<td>&nbsp;</td>
<tr>
<td>Multichannel pipette</td>
<td>&nbsp;</td>
<td>Rainin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Filtered pipette tips</td>
<td>&nbsp;</td>
<td>Rainin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5ml Eppendorf tubes Syringe Syinge filter (0.45-&mu;m)</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge tubes</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture incubator at 5% CO2</td>
<td>&nbsp;</td>
<td>Coulter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Beckman SW41T.I rotor</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Beckman ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>&nbsp;</td>
<td>Coulter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FACS Array</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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lentivirus production

RNA 간섭 (RNAi)은 살아있는 세포에서 유전자 입을의 시스템입니다. RNAi에 유전자가 상동 짧은 간섭 RNAS을 순서대로 (siRNA) 포스트 transcriptional 상태에서 침묵하고 있습니다. 짧은 헤어핀 RNAS, siRNA에 엽 성의 전구 물질은 세포 유형의 다양한 RNAi 수있게 lentivirus을 사용하여 표현할 수있다. 같은 인간 면역 결핍 바이러스로 Lentiviruses는, 세포를 분리 및 비 분리를 모두 감염을 수 있습니다. 우리는 어떤 패키지 lentiviruses에 절차를 설명합니다. 포장은 DNA 벡터에서 관할 바이러스의 준비를 말합니다. Lentiviral 벡터 생산 시스템은 자연의 바이러스 게놈은 개인 도우미 플라스미드 구조로 분할되어 &#39;분할&#39;시스템을 기반으로합니다. 네 별도의 벡터로 다른 바이러스 요소의 분할은 lentiviral 게놈의 우발적인 재조합에 의한 복제 가능 바이러스를 생성의 위험을 감소. 여기, 관심을 세 포장 벡터의 shRNA (P - VSVG, pRSV, pMDL)를 포함하는 벡터가 transiently 인간의 293 세포에 transfected 수 있습니다. 최소한 48 시간 잠복기 후, 바이러스가 포함된 뜨는은 수확과 집중되어 있습니다. 마지막으로, 바이러스 titer는 기자 (형광) 흐름 cytometer와 표현에 의해 결정됩니다.

Watch this Scientific Journal Video about Lentivirus Production at JoVE.com

Lentivirus Production

lentiviruses을 만들려면, DNA 벡터는 인간의 293 세포에 transfected 수 있습니다. 수확 후 뜨는을 집중, 바이러스 titer는 흐름 cytometer로 형광 표현식에 의해 결정됩니다.

Lentivirus 제작

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

Research

JoVE Journal

Biology

65.4K 조회수.  University of California, San Francisco - UCSF. lentiviruses을 만들려면, DNA 벡터는 인간의 293 세포에 transfected 수 있습니다. 수확 후 뜨는을 집중, 바이러스 titer는 흐름 cytometer로 형광 표현식에 의해 결정됩니다.

비디오: Lentivirus Production

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

전체 게놈 배열 비교 게놈 하이브 리다이 제이션의 기술의 데모

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

연구

생물학

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

대형 삽입 환경 게놈 라이브러리 제작

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Stemgent 인간 TF의 Lentivirus와 인간의 섬유아 세포를 프로그래밍하여 유도 Pluripotent 줄기 세포의 생성 세트

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

KLRG1 Tetramer의 생산을위한 프로토콜

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

유전과 Epigenetic 분석을위한 파라핀 임베디드 재료에서 DNA 추출

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

향상된 Biosafety에 대한 고분자 - complexed Ecotropic Lentivirus과 인간의 세포의 형질 도입

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBL에서 MultiBac 단백질 복잡한 생산 플랫폼

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

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 ...

Production of Double-stranded DNA Ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis.
Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of &#39;minicircle&#39; DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kinetics - they thus require lower amounts of cationic surfactants for effective transfection of target cells.
We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable.

The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings.

이중 가닥 DNA Ministrings의 생산

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

최적의 Lentivirus 생산 및 세포 배양 조건에 필요한 성공적으로 형질 도입 기본 인간 기관지 상피 세포

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...