s******y
发帖数: 28562 | 1 手头上有一堆retroviral vector (pBabe and pQC, PsR, etc.) 一直都是
用retroviral packaging system (GAG/PoL + VsvG)
最近想用lentiviral system,以前从来没有用过，所以想问一个白痴问题，
就是能不能直接用以前的vector装到lentiviral packaging system里面?
随便看了看lentiviral 的说明书，貌似好像没有问题？ |
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s******y
发帖数: 28562 | 2 Retroviral vector 只感染那些分裂中的细胞，对于不分裂或者不怎么分裂
的细胞（比方说原代细胞）不太好使。我有很多做在retroviral vector 里面
的蛋白，如果要一一转移到lentiviral vector 里挺花时间的。
不过听大伙的说法，这个貌似是没有办法的。。。
粒。 |
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r******g
发帖数: 600 | 3 鄙人在用 一个9kb的retroviral的plasmid 克隆一个transgene，transgene很小，大概
只有300bp～
在petri dish上面每次colony数量就不多
miniprep culture经常完全没有grow～
据说retroviral的vector相对比较大，总会有各种问题，请问大家有没有遇到过类似问
题？
Thanks |
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t**l
发帖数: 109 | 4 第一个问题是： 这个retroviral 系统会不会导致random integration?
如果有random integration,这个问题是不是已经被忽略了？
第二个问题是： 这个shRNA 是不是永久表达? drug selection gene 需不需要加，如
果有GFP在这个construct里面，能不能FACS出来GFP+ 细胞，是不是就不需要drug
selection gene.
第三个问题： 当初的viral transduction效率有多高？ 在ES细胞上如何。
最后一个问题是： 如果做screen, 这个barcode 当初在做library的时候是不是唯一的。 |
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l**********1
发帖数: 5204 | 5 that slide ppt file didn’t include after 2010 fall to now papers,
pls check,
200 times of ordinary retroviral vector that High-titre retroviral vector
system
Wu C, Lu Y.
High-titre retroviral vector system for efficient gene delivery into human
and mouse cells of haematopoietic and lymphocytic lineages.
J Gen Virol. 2010 Aug;91: 1909-18.
Abstract
Genetically modified cells of haematopoietic and lymphocytic lineages could
provide potentially curative treatments for a wide range of inherited and
... 阅读全帖 |
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L******r
发帖数: 141 | 6 All the materials were taken from Administrative Decisions (Aliens with Extraordinary Ability/ Outstanding Professors and Researchers). 所以这些话是权威!!!
I downloaded All the AAO decisions for the past three years. I read one by one for a total number of more than 200 decisions. I took good paragraphs from recommendation letters which IOs thought have met criterion of contribution. Because the works were from PDF files, I had to reformat them into word file.
It took me many hours to do the work and I... 阅读全帖 |
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a*******s
发帖数: 117 | 7 自然发生的跨远距离物种基因转移举例：
病毒基因转移到哺乳动物，包括人类
PNAS(2012)
Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral
envelope gene involved in placentation and conserved in Carnivora
ABSTRACT
Syncytins are envelope protein genes of retroviral origin that have been
captured for a function in placentation. Two such genes have already
been identified in simians, two distinct, unrelated genes have been
identified in Muridae, and a fifth gene has been identified in the
rabbit. Here, we searched for s... 阅读全帖 |
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j********r
发帖数: 156 | 8 最近一直在尝试overexpress a gene in primary epithelial cells, but always
failed to get decent level of expression。望版上诸位帮看一下。
我用的是一个类似与pBabe的retroviral vector, the gene was tagged with GFP or
Flag on the c-terminal. The target cells were infected twice and then
subjected to antibiotic selection (blasticidin in this case). 用anti-
Flag做western, 勉强能看到带，如果用the ab against itself, 可以看到exogenous和
endogeneous的差不多 （the endogenous level is already very low, which is
also the reason that we hope to increase it to ... 阅读全帖 |
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i*****i
发帖数: 154 | 9 老大要找的质粒应该是叫STEMCCA
Stem Cells. 2009 Mar;27(3):543-9.
Induced pluripotent stem cell generation using a single lentiviral stem cell
cassette.
Sommer CA, Stadtfeld M, Murphy GJ, Hochedlinger K, Kotton DN, Mostoslavsky G.
Abstract
Induced pluripotent stem (iPS) cells can be generated using retroviral
vectors expressing Oct4, Klf4, Sox2, and cMyc. Most prior studies have
required multiple retroviral vectors for reprogramming, resulting in high
numbers of genomic integrations in iPS cells and limiting... 阅读全帖 |
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l*****7
发帖数: 8463 | 10 http://www.nature.com/news/the-unsung-heroes-of-crispr-1.20272?WT.mc_id=TWT_NatureNews
The unsung heroes of CRISPR
The soaring popularity of gene editing has made celebrities of the principal
investigators who pioneered the field — but their graduate students and
postdocs are often overlooked.
Heidi Ledford20 July 2016
When Blake Wiedenheft started studying microbes, his work was both remote
and obscure.
He spent his PhD sampling hot springs in Yellowstone National Park, then
created artificial ... 阅读全帖 |
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w*********g
发帖数: 30882 | 11 基因武器比核弹更可怕
综合《看世界》、《扬州日报）消息，2003年发生的非典疫情，全球确诊8437个病
例，死亡813人。非典病毒若复制成基因武器，将是人类的灾难。这种忧虑绝非祀人忧
天。2006-2007年，以色列科学家奥登里奇将携有名为“以色列急性麻痹病毒”的实验
用蜜蜂带到美国，少数蜜蜂意外逃跑，导致美国35个州的家养蜜蜂一起远走高飞，有些
养蜂场一小时内“出走”的蜜蜂就占到70％以上，造成北美农林产业140亿美元损失。
这种病毒正是奥登里奇研制的基因武器之一！
基因武器有3种类型
基因武器又称遗传工程武器或DNA武器，是采用遗传工程技术，用工程设计的办法
，通过基因重组，在一些致病细菌或病毒中接入能对抗普通疫苗或药物的基因，或者在
一些本来不会致病的微生物体内接人致病基因而制造成生物武器。它能改变非致病微生
物的遗传物质，使其产生具有显著抗药性的致病菌，利用人种生化特征上的差异，使这
种致病菌只对具有特定遗传特征的人们产生致病作用，从而有选择地消灭敌方有生力量。
目前，世界基因武器的发展... 阅读全帖 |
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w*********g
发帖数: 30882 | 12
榨取型强调的是没有实现法治化、没有言论自由、政府腐败、极权专制的情况下，统治
者通过政治权力直接夺取。美国根本不是这样。
实际上，美国在过去几十年实现了一系列的颠覆性创新，如下
Internet, broadband, WWW (browser and html)
PC/laptop computers
Mobile phones
E-mail
DNA testing and sequencing/Human genome mapping
Magnetic Resonance Imaging (MRI)
Microprocessors
Fiber optics
Office software (spreadsheets, word processors)
Non-invasive laser/robotic surgery (laparoscopy)
Open source software and services (e.g., Linux, Wikipedia)
Light emitting diodes
Liquid crystal display (LCD)
GPS syst... 阅读全帖 |
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r***y
发帖数: 4379 | 13 官网写着是dr xxx
原文
Robert R. Redfield, MD
Robert R. Redfield, MD, is the 18th Director of the Centers for Disease
Control and Prevention and Administrator of the Agency for Toxic Substances
and Disease Registry. He has been a public health leader actively engaged in
clinical research and clinical care of chronic human viral infections and
infectious diseases, especially HIV, for more than 30 years.
He served as the founding director of the Department of Retroviral Research
within the U.S. Military’s... 阅读全帖 |
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m******1
发帖数: 19713 | 14 HIV: Early Treatment Could Reduce Partner Transmission
Healthy HIV-positive people taking oral retroviral medications early in
their infection reduced giving the virus to sexual partners by 96%.
By Advocate.com Editors
MEN COUPLE LOVE MARRIAGE MONOGAMY X390 (PHOTOS) | ADVOCATE.COM
HIV-positive people with healthy immune systems, who started taking oral
antiretroviral medicines early in their infection reduced the risk of
transmitting the virus to their sexual partners by 96%, according to new
da... 阅读全帖 |
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D**S
发帖数: 24887 | 15 http://news.yahoo.com/s/yblog_thelookout/20110603/us_yblog_thel
Since HIV was discovered 30 years ago this week, 30 million people have died
from the disease, and it continues to spread at the rate of 7,000 people
per day globally, the UN says.
There's not much good news when it comes to this devastating virus. But that
is perhaps why the story of the man scientists call the "Berlin patient" is
so remarkable and has generated so much excitement among the HIV advocacy
community.
Timothy Ray Brown... 阅读全帖 |
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y*****g
发帖数: 1822 | 16
“China is seen internationally as a late-responder to the Aids crisis
and this is a clear example of what can be achieved.” The authors of the
study concede that despite the improvement, much more work needs to be done
in China to improve Aids care.
“Given the size of the country, and the geographical spread of individuals
with HIV…China’s treatment coverage is remarkable,” said Professor Fujie
Zhang from the National Centre for AIDS Control, “But it is far from the
goal of complete covera... 阅读全帖 |
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J*V
发帖数: 3150 | 17 基因武器已成为现实。
直言了，2012-03-13 | 2012-03-14 22:05:20。
http://zhiyanle.blog.hexun.com/73735052_d.html
两会上，有代表阐明了转基因技术的危害风险、说明了它可用来做杀伤武器，由此而提
出了中国需要建立“生物国防”体系的建议，其中包括对转基因技术产品的严格检验、
监控乃至限制或禁止等措施的建议。该建议妨碍了转基因既得利益集团的商业利益，于
是，他们雇用的枪手文人接连出来，说转基因技术可制造杀伤武器的说法是“危言耸听
”云云。其实，在美国，用转基因技术搞基因武器不但已经是现实了，而且已经开始公
开征集相关国防用品设计了。故事很多，下面就说最近发生的一个故事。
2012年开始，美国国防系统的“达尔帕”（DARPA）部门公开招标“蛋白折叠”软件设
计，项目名称是“FOLD-IT”（直译：折叠它）。所谓“蛋白折叠”，就是根据某基因
结构而形成某蛋白的过程；该蛋白发挥功能可使某生命体幸存发育或残疾死亡。要实现
某种预期的蛋白折叠，就先要实现某种基因结构。到目前，基因结构设计包括DNA-水平
和RNA-水平的两种设... 阅读全帖 |
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l****z
发帖数: 29846 | 18 by Rick aka Mr. Brutally Honest
Surely there were times he infuriated me and yes, I questioned during those
times who he really was... but given who's been sitting in the Oval Office
since he departed, and given stories like this one, yes, I miss him... and
if you don't, then it would be dishonest of me to not question the state of
your soul:
Former President George W. Bush has kept a relatively low profile in the
United States since leaving office. But in Africa this week he is publicly
pr... 阅读全帖 |
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m*********a
发帖数: 3299 | 19 Jews .2% of world population, 20% of all Nobel prices, 40% of USA nobel
price, won more than 160 nobels.
All Asian are junk compare to Jewish people in your standard.
Literature[edit source | edit]
Year Laureate Country Rationale
1927 Bergson-Nobel-photo.jpg Henri Bergson[9][10] France "in
recognition of his rich and vitalizing ideas and the brilliant skill with
which they have been presented"[11]
1958 Boris Pasternak cropped.jpg Boris Pasternak[9][10] Sov... 阅读全帖 |
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q****2
发帖数: 208 | 21 【 以下文字转载自 Biology 讨论区 】
发信人: qq3702 (I shall be your eyes!), 信区: Biology
标 题: 转一个工作机会（Research Associate），有兴趣可以联系下
发信站: BBS 未名空间站 (Tue Feb 13 17:25:24 2018, 美东)
转一个工作机会（Research Associate），有兴趣可以联系下。
Are you passionate about joining a fast-paced startup in the breakthrough
field of cancer immunotherapy? Our research team in Los Angeles is seeking a
highly motivated individual to perform hands-on laboratory work. TCRCure is
a one-year-old biotech startup with a global focus to provide personalize... 阅读全帖 |
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n*******y
发帖数: 3337 | 22 Today, 12:32 pm #16
MilesGator
Join Date: Sep 2012
Posts: 50
If it was a psuedo FR (i.e. just a phone call to verify that it was the
cardholder who actually bought it), then saying it's for drugs is no problem.
But if it were to escalate to a real FR from Amex, wouldn't they look
closely at the line-item receipts and see that the charge wasn't for Anti-
Retrovirals?
If you lie at first then it may just go away and everyone is happy. But if
you lie at first and it somehow gets escalated... 阅读全帖 |
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z****l
发帖数: 5282 | 23 ☆─────────────────────────────────────☆
newjersey (NJ furniture dealer) 于 (Mon Jan 28 13:07:10 2013, 美东) 提到:
Today, 11:22 am #1
noturbizniss
Join Date: Sep 2009
Posts: 13
Financial Review with CVS 1K purchases
Searched for a master FR thread but couldn't find it, or this question
addressed...
If asked by amex about multiple CVS purchases of 1007 what's the best course
of action?
☆─────────────────────────────────────☆
newjersey (NJ furniture dealer) 于 (Mon Jan 28 13:07:46 2013... 阅读全帖 |
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y****1
发帖数: 111 | 24 明天后天两天，11am-7pm，在哈佛广场有吉尼斯世界纪录“史上最大钱包”展览，我们
的CHARITY受邀做宣传，欢迎走过路过的过来看看。你也可以将零钱捐入世界最大的钱
包内，我们的SPONSOR将1：1 match你的捐助。
http://www.patskids.org/
在中国资助45个艾滋病儿童，所筹款项全部用于中国，在美工作人员为100%志愿者。
The Pediatric HIV/AIDS Treatment Support Project (PATS) was established in
2007 in response to an urgent and on-going situation: the care of HIV-
positive children in rural China. In parternship with the AIDS Orphan
Salvation Association (AOS) of China, PATS aims to promote the health of
children affected by HIV/AIDS.
... 阅读全帖 |
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t******g
发帖数: 17520 | 25 Using a game called Foldit, gamers build enzyme models that may be crucial
to understanding how many diseases do their damage. Scientists couldn’t
solve the crystal structure of the M-PMV retroviral protease for more than
10 years. Gamers solved it in 10 days. Zoran Popovic is one of the
conceptual designers of Foldit - a kind of 3 dimensional Tetris.
http://www.msnbc.msn.com/id/44668197/ns/local_news-new_orleans_ |
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p********3
发帖数: 5750 | 26 50， 70， 71； 大家注意一下和这几篇文献相关的话题。 另外， 73-75我本人没有看，确实是谈的印度药材， 我在查阅参考文献的时候在搜索Chinese Medicine的时候， 显示有这些文献，我本人没有亲自看这些文献， 也许其中有讲中药的地方。不过这不影响我要表达的意思。
慢慢看：
INTRODUCTION — Plants have been used for medicinal purposes for thousands
of years. All major cultures including Native American, European, South
American, Asian, and African cultures have used botanicals for healing
purposes. As an example, saw palmetto was used for urinary symptoms in men
from Egypt in the 15th century BC [1]. Hippocrates documented... 阅读全帖 |
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m*******d
发帖数: 4 | 27
To study NSC differentiation, GFP and differentiation maker can be used in
vitro, which is relatively easy. In vivo differentiation
studies usually adopt 2 strategies: transplantation or retroviral injection
to trace the progenies of NSC. People have done that,
showing that different cell types are generated, but it's really hard to say
these different progenies are from the same SINGLE
ancestor. Till now, I think there is still NO convicing evidence to demostra
te NSC existence in vivo. Some p |
|
F*****8
发帖数: 243 | 28 这个我知道， 用 retroviral vector 当然不一样。 我想知道的是用纯 DNA 转染能达
到多高的整合效率？
谢谢 |
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n********k
发帖数: 2818 | 29 May I ask for ur protocol, thanks...BTW, by insertional KO, u mean using
retroviral like that, right?
40 |
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n********k
发帖数: 2818 | 30 unless in special occasions/needed/desired(say u don't want the high
expression etc), what's the point making stable cell lines if you use
retroviral approach...I(many if not most) rarely keep them as stable lines..
..Nolan's lab is an excellent place for viral stuff...Yes, u can keep them
as stable cell lines,but you would have to take much precautions...single
clones is an old old(updated too) approach...of course u cannot do one
clones, u need at least dozens of clones intially and then narro |
|
n********k
发帖数: 2818 | 31 BTW, the way u are doing is likely fine...within several passage is fine or
as long as they are well-chaterized, it is fine...but is it that hard to go
retroviral and treat them as transient approach...I rarely keep any stable
cell lines now...And for many studies, it is impossible or not right to keep
stable cell lines because the manipulation cause them to change...for
example differentiation...so u are screening against the correct phenotypes.
..anyway, nothing is absolute and it all depends. |
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n********k
发帖数: 2818 | 32 likely yes, but never compare that(besides the normal silencing, retroviral
has extra silencing effects)...but for single clones, u are picking those
with good/reasonable expressions, so there isn't a real issue in term of
promoter silencing because you already exclude those when u pick the clones.. |
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n********k
发帖数: 2818 | 33 don't know, I haven't followed closely lately with this as I do mostly in
vivo now...in vitro is just for confirmation or mechanistic probing...for ur
case, it is likely less a problem...I am more talking about in term of
phenotypical changes...BTW, as I said earlier, I don't see why you have to
use stable cell lines if with retroviral... |
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y*******i
发帖数: 96 | 34 刚来一个实验室做博后，以前的博后走了，也没有具体的protocol,我们有pBABE和
pBABE-ras retroviral vector. 这些载体是可以直接用fugene-6转染到人的细胞（比
如MCF-7)中，还是需要先把这些载体包裹到virus中，然后再感染细胞．目的是要看到
ras-induced senescence.好像文献中都是需要packing,然后再感染细胞．
多谢 |
|
X***n
发帖数: 366 | 35 DH5a or Invitrogen Stbl3? It says Stbl3 is better for retroviral vector. |
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i******0
发帖数: 17 | 36 用retrovirus deliver transgene into NIH3T3 cells. 以前都用一次infection, 然
后就做selection. 蛋白表达不太高. 请问如果我infect 两次, 或者是三次, 会增加蛋
白的表达量吗? 若要这麽做, 每次infection 之间需要做selection 吗?
请高手指教, 谢了. |
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y**y
发帖数: 36 | 37 一次感染6-12h就可以了，所以如果你以前是O/N，现在可以分成2-4次，应该够了吧 |
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i*********t
发帖数: 78 | 38 多次infection 不如浓缩病毒， 用high MOI 一次infection, 确保每个细胞至少插入
一个copy. |
|
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j*****q
发帖数: 82 | 40 lz如果你能保证second infection插入的copy是在第一次infection的同一个位置，还
是有机会提高protein level的。double transfection有人用来提高protein产量的。 |
|
m******5
发帖数: 1383 | 41 Who knows if this cell line a DTA resistant cell line??
Thanks alot!!!!
I didn't find it in the protocol |
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i*****g
发帖数: 11893 | 42 我是有人用genetic deletion 清理了外源表达，才开始信iPS
这个retroviral iPS 本身就含有强制表达的基因，有问题很正常
这个EiPS 可能是incomplete reprogramming，
前面还有CNS文献，说retro iPS含有很多gene deletion/duplication，这个也好理解
，细胞是被强制进入cell cycle
iPS是个很有前途的领域，对我而言，最重要的意义不只是ESC- iPS，更重要的意义是
：某种细胞可以用外来表达强制变成另外一种细胞，而且在撤出外来控制之后，能稳定
维持那个‘态’。 |
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H****s
发帖数: 301 | 43 对于puromycin抗性的克隆筛选来说，这种情况是非常普遍的。看看你的载体吧，如果
标靶基因和puromycin抗性是由不同的启动子驱动的，那么你运气不太好，需要多筛选
克隆。主要有两种解释：（1）lentiviral/retroviral载体介导的异源表达，很普遍的
一个现象是整合到基因组的序列被flanking genomic sequences灭活。具体怎么回事，
现在还不太好解释。（2）低表达或者是不表达的细胞outgrow表达标靶蛋白的细胞，所
以，几代以后，你基本上丧失标靶蛋白的表达。
有两个建议：
（1）。使用抗性基因和标靶基因被同一启动子驱动的载体。标靶基因和抗性基因中间
有IRES。这样，产生抗性的细胞系必然表达标靶蛋白。如果被flanking genomic
sequences灭活的话，细胞会死亡，因为抗性丢失。
（2）。如果筛选到合适的细胞系，在前面几代要多冻存细胞。这样在后面的实验中你
不会丢失辛辛苦苦得来的细胞系。 |
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M*****n
发帖数: 16729 | 44 retroviral insertion是老路子，但是也有limitation.
其实这就是NIH到中国去骗钱。 |
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p****e
发帖数: 1336 | 45 nature structural & molecular biology advance online publication
Crystal structure of a monomeric retroviral protease solved by protein
folding game players |
|
j****x
发帖数: 1704 | 46 新一代lentivector中很多都带这个SV40 ori，在293T细胞中支持episomal DNA
replication，理论上对提高病毒titer肯定有好处。retroviral vector中最早的报道
是可以提高病毒滴度up to 2 logs，lentivector上的数据我不是太清楚 |
|
i*****i
发帖数: 154 | 47 Nat Biotechnol. 2004 May;22(5):589-94. Epub 2004 Apr 4.
Correction of multi-gene deficiency in vivo using a single 'self-cleaving'
2A peptide-based retroviral vector.
Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF, Vignali
DA. |
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D*******8
发帖数: 4 | 48 Please don’t dig me out. I am writing this out of my conscience as a
scientist. We are using, largely, the tax payers’ money to explore
mechanisms underlying diseases that we don’t have a cure, yet. We are doing
this in the hope that little bit knowledge can accumulate till one day we
can find that magic bullet. However, there are people out there, their
purpose is to use these money to achieve their personal fames, sometimes
international ones, totally disregarding our original noble cause. I... 阅读全帖 |
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c*****u
发帖数: 357 | 49 我想构建表达蛋白的stable cell line,之前已经构建了MCF10，现在用同样的方法做
infection，结果一加puromycin(2ug/ul)，MCF7细胞基本全死了，empty vector也是。
侵染时我一般加8ug/ul的polybrene。MCF7属于luminal, 听说luminal很难做侵染，不
知道有经验的朋友们有什么建议，谢谢！ |
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