C**S
发帖数: 522 | 1 pBABE是Retroviral的
second
ml
selection |
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l**********1
发帖数: 5204 | 2 LZ can try this:
Posted: 3/13/2012
Position Title :
Research associate
Available: 07/01/2012
Qualifications:
Interested candidates must hold a Ph.D. or MD degree, have 5 years of post-
doc experience and should have expertise in one or more of the following
fields: regulation of gene expression, transcription factors, chromatin
immune precipitation and Ch-IP Seq analysis, gene expression arrays,
bioinformatic assessment of high throughput sequencing and genomic data,
knock-out and transgenic mou... 阅读全帖 |
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d*p
发帖数: 534 | 3 I ever made a mistake, pack retro vector with lenti system. It failed. So I'
ll say NO. |
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s******y
发帖数: 28562 | 4 如果是这样的话那可就太麻烦了。我可不想把我好不容易克隆到retro里面的东西
又倒腾克隆一次。。。
I' |
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r***e
发帖数: 2539 | 5 不行。
另外包装lenti也要注意，尽量用第三代的。
切记不能把不同generation的vector和pkg mix混用，
有的公司为了规避patent，还在出售第二代的vector，比如说Openbiosystem有名的pLK
O shRNA vector，和Thermo的部分产品。
以前有实验室出过事故，包装出不安全的病毒。 |
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i*****i
发帖数: 154 | 6 试过，效率确实很低。
为什么不在Addgene买一个包装的质粒pUMVC (Plasmid 8449),65刀就可以搞定了。如果
你参加今年的AACR的会的话儿，可以在Addgene拿一个免费的coupon，会赠送一个质粒。
或者买一个phenix cell，效率很高。 |
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i*****i
发帖数: 154 | 9 二代和三代的差别，在packaging system上，由二代的Gag/pol/Rev + VSVG，变成了
Gag/pol+Rev+VSVG，两质粒变成三质粒。病毒的编码区的进一步分割，分离，可以降低
重组野生病毒（有自主复制和感染能力）的可能性。
比较典型的二代是pspax2+pMD2.G 以及pcmv dvpr 8.2+pCMV VSVG。
而比较典型的三代是pMDLg/pRRE+pRSV-Rev+pMD2.G, 以及Invitrogen的包装系统，都是
三代。
其实clontech的包装系统应该是第四代了，虽然这个系统采用了旧的驱动系统，但是对
整个病毒蛋白的编码区做了进一步的split，并引入了tet调控，所以，应该是更安全一
些。但是共转染7个质粒的效率，显然不如三个。所以，第二代系统仍然是titer最高的
最有效的系统。 |
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s******y
发帖数: 28562 | 10 谢谢！
我从来没有用过lenti, 所以都不知道有这种差别。受教了。
另外，为什么retro vector 不能装进lenti 系统里面呢？难道他们的包装
信号差的很远么？
我一直误认为lenti就是多了一个强行进入细胞核的蛋白，这么看来还是有
别的差别？ |
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n********k
发帖数: 2818 | 11 no, it seems u need to do a better homework:))...the packing systems for
lenti and retro are the same except lenti need one extra...the difference is
lenti vector has the bosai element which determine the nuclear import...
that's why lenti can infect both dividing and non-dividing cells...frankly,
I haven't seen any retro which doesn't infect non-dividing at all... |
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i*****i
发帖数: 154 | 12 第二点，在表达载体上，第二代表达载体采用野生型的 HIV的LTR，而第三代载体采用
了chimeric的LTR，所以你经常会看到第三代表达载体的LTR写成CMV/LTR或者RSV/LTR用
来表示这种杂合LTR结构。
无论是lenti还是retro，LTR都既是promoter又是enhancer，比较一下lenti和retro的
LTR差别还是非常大的。
其实我们试过，lenti的包装系统可以包装retro的，而retro的也能包装lenti的表达载
体，只是效率非常非常的低，没有实际的可操作的意义，还不如直接用pcDNA3.1感染呢。
lenti里的特殊结构
WPRE (Woodchuck
Posttranscriptional Regulatory Element) from the woodchuck hepatitis
virus, increases transgene expression.
cPPT (central Polypurine
Tract) from the HIV-1 integrase gene, increases the copy number o... 阅读全帖 |
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l**********1
发帖数: 5204 | 13 还不把你楼上的叫inanxx 啥的招同济去
如其CNS 记录比你还多和还牛
要不你当二当家的 让她/他当大当家的得了
就像林冲刚上了梁山那会儿...
有这气魄吗?
is
, |
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m******n
发帖数: 121 | 14 问一下，可以用rt-pcr测ltr的方法检测lenti的滴度吗？另外这个方法怎么样呀？主要
是我有一个lenti没有gfp，然后p24的试剂盒么又太贵，所以在考虑用rt-pcr测滴度。
。。。。 |
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f*******e
发帖数: 354 | 15 pbabe是retroviral的吧，LTR表达量其实也很高 |
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d**b
发帖数: 1286 | 16 A postdoctoral position is available to study the functions and molecular
mechanisms of microRNA control in lymphocyte development, immune responses,
immune tolerance, autoimmune diseases, and lymphoma. For more information,
see Cell (2007) 131:146-59, Nature Immunology (2008) 9:405-14, Cell (2009)
136:26-36.
Requirements:
Highly motivated PhD, MD, or MD/PhD with a solid background in immunology.
Working experience in the following areas is desired:
The mouse immune system
immune tolerance;
Mous... 阅读全帖 |
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p****z
发帖数: 31 | 17 查了一些文献，很多都写得不是很明确。想用pBabe-based的virus去infect THP-1和
MOLT-3这两个细胞系。
在sigma看到一个，其中有一步是加入virus之后离心半小时，之后就立刻把上清去掉，
不甚理解，所以不敢用。
谢谢大家！ |
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n********k
发帖数: 2818 | 18 an easiest way is to just try it with GFP-virus and the chance is one need
to optimize or repeat the procedure anyway...I work with adherent cells but
I typically do infection in suspension in small volume, never bother to wash
off the virus etc unless there is any specific reason for it...BTW, if one
knows the principles of viral infections, one might understand more of an
infection protocol---why this not that... |
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n********k
发帖数: 2818 | 19 I doubt you have done the readings...In short, yes and no...For some, the
lenti packging can be used for Retro(pbabe). So for dividing cells, doesn't
matter much for the infection/integration; However, for non-dividing cells,
presumably no big difference with infection but a huge difference with
integration step(nuclear entry step--which is essentially the key difference
between the two)...So essentially western doesn't tell you much here...
unless they stablize.
BTW, contrast to the common beli... 阅读全帖 |
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j******i
发帖数: 939 | 21 万能的买买提啊，有没有人恰好可以下载current gene therapy的文章呢？！急需两篇
文章：
NO1: Editorial (Hot Topic: Vectorizing mRNA and Proteins) Pp.345-346
Axel Schambach and Christopher Baum
NO2: Retroviral Protein Transfer: Falling Apart to Make an Impact Pp.389-409
Tobias Maetzig, Christopher Baum and Axel Schambach
如能下载，麻烦发送到我的邮箱：y***[email protected]
谢谢！ |
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l**********1
发帖数: 5204 | 22 RE LZ
here you go
Plasmid Information
Toolbox-No. 114
Functional Description Retroviral expression vector derives from pMIG;
the GFP of the original IRES-GFP casette is exchanged with the enhanced cyan
flourescent protein. . The vector allows cloning of genes of interest on
the N-terminus of IRES for co-expression with CFP in cells.
web link:
http://www.bioss.uni-freiburg.de/toolbox/products.php?PL-62
then pls send mail to German side for it:
link:
http://www.bioss.uni-freiburg.de/toolbox/... 阅读全帖 |
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C***2
发帖数: 62 | 23 Hey, I have read several articles using 2A to generate transgenic mouse,
please refer the following articles( I just copy the titles, it is very easy
to search fulltext if you are interesting):
1.High Cleavage Efficiency of a 2A Peptide Derived from
Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and
Mice
2.Correction of multi-gene deficiency in vivo using a
single ‘self-cleaving’ 2A peptide–based retroviral vector
3.Faithful Expression of Multiple Proteins via 2A-Peptide Self-Processing:... 阅读全帖 |
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a******a
发帖数: 283 | 24 ask a question in this thread: regarding genomic DNA purification. I will be
doing about the same project -- qPCR genomic DNA for the amount of gene
inserts after transfecting the cells with retroviral vectors.
My plan is to transfect the cell culture on day 1; then on day3, harvest
cells and collect genomic DNA. at this point, how to eliminate the plasmids
present in the cells (if there is still any left without incorporating in
the genome)? would centrifugation alone enough to get rid of most ... 阅读全帖 |
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r**o
发帖数: 212 | 25 FASEB J. 2012 Aug;26(8):3140-7. doi: 10.1096/fj.11-198515. Epub 2012 Apr 24.
MicroRNA-31 targets FIH-1 to positively regulate corneal epithelial glycogen
metabolism.
Peng H, Hamanaka RB, Katsnelson J, Hao LL, Yang W, Chandel NS, Lavker RM.
Corneal epithelium relies on abundant glycogen stores as its primary energy
source. MicroRNA-31 (miR-31), a corneal epithelial-preferred miRNA,
negatively regulates factor inhibiting hypoxia-inducible factor-1 (FIH-1).
Since HIF-1α is involved in anaerobic ene... 阅读全帖 |
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m*****u
发帖数: 15526 | 26 谢谢大侠。能否提供进一步信息？用的哪个retroviral vector？用什么包装细胞系？
用不用PCL－Eco之类helper plasmid？ |
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m*****u
发帖数: 15526 | 27 没找到retroviral vector.都是lentiviral vector.用lenti的话转染效率跟retro比哪
个高？对T细胞而言
.
something |
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m*****u
发帖数: 15526 | 28 谢谢大侠。能否提供进一步信息？用的哪个retroviral vector？用什么包装细胞系？
用不用PCL－Eco之类helper plasmid？ |
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m*****u
发帖数: 15526 | 29 没找到retroviral vector.都是lentiviral vector.用lenti的话转染效率跟retro比哪
个高？对T细胞而言
.
something |
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M****1
发帖数: 2 | 30 最近用PHEONIX CELL 产retroviral， transduction SHRNA-GFP到mda231细胞老是效率
很低<1%。换了细胞线，加了3ML病毒 用了10UG/ML POLYBRENE，；连转了2-3次，不同
的CONFLUENCY, 还是很低，其他细胞MCF7还行 20-30% 或 30-40%不等。
是不是这个细胞线很TOUGH?还是VECTOR的问题？还是有其他方法？ 请问有没有经验的
大虾，请指教。多谢先！ |
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x*****n
发帖数: 825 | 31 想和GFP一起做coinfection，不知道有没有好心人share一下。可以提供的好心人请站
内联系我，非常非常感谢！ |
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a**********4
发帖数: 15 | 32 版上各位大侠，我现在实验室刚开始从事IPS细胞的课题，以前实验室从来没人做过，
我查了一些文献，发现现在是不是都用一个Lentivral vecotor上挂4个gene来诱导IPS
细胞？
山中申弥的用4个retroviral的办法是不是已经不用了？
如果哪位大侠有好的protocol，请发站内信箱，5个包子的奖励。 |
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c******r
发帖数: 3778 | 33
//会
//是
//是
drug selection gene 需不需要加，
//一般drug selection 比较方便长期保持shRNA表达。
如
//可以。但是如果长期培养，会导致逐步失去shRNA的表达，也失去GFP表达。
//depends，自己测试就知道了。
的。
//看barcode怎么设计的。一般应该是的。 |
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C**S
发帖数: 522 | 34 刚做了一个retroviral plasmid，5k的载体，6k的基因，大概1：1比例，室温连接6个
小时，转化top10，筛了几个克隆，全都对。不知道你的问题出在哪里。 |
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C**S
发帖数: 522 | 35 我最近也在装一个载体，把一个5k的基因装到pEGFP－C1载体再把GFP fusion蛋白装到
pBabe retroviral里。酶切测序都对，就是转到293T细胞里只能看到极少数细胞GFP阳
性。真的是因为基因太大了？谢谢。 |
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z*t
发帖数: 863 | 36 你是说这段么“A disadvantage of this type of double-copy vector is that the
RNA form of the vector genome contains two copies of the cDNA, and since the
size of the vector RNA is limited to about 10 kb, the size of the cDNA that
can be accommodated is about half of that possible in other retroviral
vectors.”
好像说的是因为retrovirus要搞two copy of RNA，而two copy加起来的总共最多可以
装10kb，所以单个cDNA不能超过5kb?
不知道目前的MSCV-GFP/neo是不是这种double copy vector
kB. |
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C**S
发帖数: 522 | 37 还有这句：
Although there appears to be no lower limit on retroviral vector size, there
do appear to be upper limits. The genome of a typical replication-competent
murine retrovirus is about 8.3 kb, whereas that of RSV, which contains src
sequences in addition to the normal complement of viral genes, is about 9.3
kb. The maximum size for a replication-competent spleen necrosis virus
vector is similar, about 10 kb (Gelinas and Temin 1986). Although this
precludes vector transmission of large genes, mo... 阅读全帖 |
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c*******r
发帖数: 54 | 38 Recent US government budgets have harmed the National Institutes of Health.
But the problems in the medical research system are deeper than just the
current budget. If you care about medical research, read this article in the
Proceedings of the National Academy of Sciences by Bruce Alberts, Marc
Kirschner, Shirley Tilghman, and Harold Varmus.
Alberts and his colleagues describe a hypercompetitive culture in science
that undermines the process of discovery.
Competition… has always been a part... 阅读全帖 |
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r******g
发帖数: 600 | 39 看到 很多大牛们出来讨论 那个克隆的问题～ 我也来征求一下大家的suggestions～
我们实验室 研究一个 mitochondrial protein的功能。 是一个mitochondrial
transmembrane protein。我的实验目的 是，克隆表达 这个蛋白的 5'-HA tag-Nter
region-Transmembrane-3' (缩写HA-Nter clone) 和 5'-HA tag-transmembrane-C-ter
region-3' (HA-Cter clone) 两个truncated protein.
clone挺简单，因为 克隆cDNA，并且，蛋白很小，只有200氨基酸。我克隆的片段也挺
小的，没有经历太多难度。我把 Nter clone 和 Cter clone 都克隆到 一个
retroviral vector，coding region后面有一个IRES-GFP。plasmid 8.6kb。
接下来,我把这个HA-Nter clone 和HA-Cter clone，用293 cell 表达（CaCl2
transfection）.
... 阅读全帖 |
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r******g
发帖数: 600 | 40 sorry，没有讲明白
2个 独立plasmids,都是retroviral plasmids
promotor是 CMV
最后克隆的两个plasmids
HA-Nter
CMV promoter--Multicloning sites--- HA tag- N-terimus+Transmembrane domain+a
few more C-ter nucleotides----- IRES------EGFP
HA-Cter
CMV promoter--Multicloning sites--- HA tag- Transmembrane domain+C-terminus-
---- IRES------EGFP |
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l*******i
发帖数: 153 | 41 这取决于你的载体的优点与A细胞在基础研究和临床应用中的重要性。
首先，与目前广泛使用的AAV，retroviral, lentiviral, transposon, episomal,
minicircal等gene delivery tools相比，你的vector有哪些优势：
转染效率如何？
安全性如何？
稳定转染的时间有多长？不会出现transgene silencing?
在deliver大于10kb的transgene cassette 有优势吗？
less immunogenicity?
操作简易程度如何？
成本控制怎样？
如果以上问题都是肯定的，特别是优于viral vector，那么你直接就上nature biotech
或nature methods吧。如果有一半问题是肯定的，那么PNAS档次的应该可以一试；如果
上述三分之一的问题是肯定的，那你就试试molecular therapy。
如果你能拿到病人的细胞，做个correction，在评价一下corrected cell的功能与
efficacy & safety profile，可以试试science tran... 阅读全帖 |
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W********g
发帖数: 610 | 42 06/29/2012 Astellas Pharma Inc. ALPMY.PK, ALPMF.PK Mirabegron (NDA) FDA
decision on Mirabegron for proposed treatment of overactive bladder
-News
06/28/2012 Bristol-Myers Squibb Co. BMY ELIQUIS (NDA) FDA decision on
ELIQUIS for prevention of stroke and systemic embolism in patients with
atrial fibrillation
06/27/2012 Arena Pharmaceutical Inc. ARNA Lorcaserin (NDA) FDA decision on
Lorcaserin for proposed treatment of obesity
06/21/2012 Repligen Corp RGEN SecreFlo... 阅读全帖 |
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