A****Z
发帖数: 52 | 1 Clontech has 3rd generation of tet-on inducible expression system but this
new system has only the general expression plasmids but no lentiviral
vector. Only the 2nd generation has inducible lentiviral vector but the 2nd
generation may not work as well as 3rd generation. My cells are difficult to
transfect, so I need the lentiviral vector to delivery the target gene to
cells. Does anybody has good Lentiviral Inducible Expression Systems?
Thanks. |
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s******y
发帖数: 28562 | 2 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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j******i
发帖数: 939 | 3 lentiviral paticles(LP) 就是光壳子 没有病毒ssRNA包装到里边 lentiviral vector
(LV)就是一般的病毒载体包含ssRNA信息。 |
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A****Z
发帖数: 52 | 4 Does anyone know if there is any good induceable lentiviral vector from any
company? Please introduce some good induceable lentiviral vectors for
cloning. Thanks. |
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j****x
发帖数: 1704 | 5 之前没留意过，近期突然发现，Clontech家的所有Lentiviral vector系统，包括其经
典的Tet-on/off/one lentivector，尽管宣传是最安全的第四代载体系统，但其中的
HIV-1 5'/3' LTR居然都是wildtype的而并非是被几乎所有其他商用载体所一致采用的
truncated/∆U3 LTR，作为self-inactivating (SIN) vector。这是件很奇怪的
事情，有哪位对Lentiviral vector比较熟悉的能讲讲吗？谢谢 |
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q*********1
发帖数: 142 | 7 跪求“Lentiviral Transduction of Immune Cells”，邮箱是[email protected]
/* */ |
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w****u
发帖数: 1078 | 8 最近打算使用RT方法作个对于人细胞的试验，不知道谁有这方面的protocol.
而且，使用这个Lentiviral Particle对人是否有危害，应该采取什么防护措施。
谢谢 |
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a********k
发帖数: 2273 | 9 sorry,没有做过lenti， 不熟悉有哪些vector可以用。有没有哪个lentiviral vector
可以同时表达两个不同的shRNA，并且有荧光marker做FACS啊？ |
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s******r
发帖数: 2876 | 10 天然的miRNA也有很多同时表达的吧，
你可以模拟他那个结构，用pTRIPZ来表达。
sorry,没有做过lenti， 不熟悉有哪些vector可以用。有没有哪个lentiviral vector
可以同时表达两个不同的shRNA，并且有荧光marker做FACS啊？ |
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w****u
发帖数: 1078 | 11 I tried retrovirus for OVER expression on murine primary T cells, there are
>60% infection. So, if lentiviral shRNA are not good for infection. Is that
better to clone this into retrovirus vector? thanks |
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L**********O
发帖数: 1761 | 12 我们是买的lentiviral vector... |
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z****g
发帖数: 3340 | 13 这个就是lentiviral vector，你有这个吗？ |
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z*******9
发帖数: 112 | 14 请问有没有人试过在Lentiviral vector里表达T7 polymerase的？可行吗？？ |
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z*t
发帖数: 863 | 15 同问，有人用过clonetech的Lentiviral Inducible Expression Systems没？价高但不
知质量如何。。。有没有其他的kit或者非tet-on/off的system可以推荐？
any |
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s******y
发帖数: 28562 | 16 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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h********n
发帖数: 4079 | 17 高人们能不能推荐一个lentiviral vector, 可以表达两个外来的gene, 比如
luciferase 和 Cre, 同时还能表达一个shRNA?
tyler jacks的那个 UbC.Luciferase.PGK.Cre 好用吗?
谢谢谢谢. |
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z******5
发帖数: 30 | 18 直接用lentiviral vector 感染细胞，病毒会整合到基因组吗？ |
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n****y
发帖数: 819 | 19 要从其他实验室弄一些被lentiviral infected 的 tumor cell line。从来没有弄个
lentivirus，不知道有什么safety地方要注意。感觉应该就像处理其他一般tumor cell
line一样就行了，应该已经没有virus了。对吗？ |
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g*********5
发帖数: 2533 | 20 wanna use shRNA (h) Lentiviral Particles and how is Santz cruz?
or other good company?
thanks |
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s*****a
发帖数: 59 | 21 没有lentiviral的经验，现在需要在难转染的细胞里做overexpression 和 knockdown
，如果有tet-inducible 的更好。细胞用的是neuroblastoma 和 primary rat neuron
。请推荐vector和protocol。不知能不能用同一个vector（比如pKLO）做OE和KD? 谢谢
！ |
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x******o
发帖数: 76 | 22 想把6kb片段连进8.5kb的lentiviral vector里，试了几次都不成功，求助一下。
我是这么做的，单酶切得到6kb insert，切胶纯化，载体单酶切，去磷酸化，切胶纯化
。1:1比例4度连接过夜，转化stbl3感受态42度45秒，recover 1小时涂版，30度过夜，
板子上长几十个斑。挑斑入LB+amp 30度摇过夜，miniprep，酶切鉴定。跑胶时，总是
得到大概2kb的片段，无论是否酶切过。拿最近的一次质粒直接跑胶结果来看，12个克
隆，9个是2kb band，2个是degraded DNA（1-2kb size），1个是5kb左右（经酶切鉴定
可能是contamination）。 现在基本怀疑是LTR重组导致片段丢失，觉得自己已经考虑
了很多可能导致LTR重组的因素，所以只能问问大伙有什么建议。 |
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h********x
发帖数: 285 | 23 请教一个问题： BMDM(bone marrow derived macrophage) 和手上的一个mouse的
immortalized macrophage cell line的lentiviral transduction效率很低，我用了８
ug/ml polybrene, 1000g 60min spin,请问大家有什么高见？ |
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z*******9
发帖数: 112 | 24 我用Lenti-X lentiviral vector （EF-1 alpha Promoter)表达我的一个viral
glycoprotein,在其C端与GFP marker之间，我加上了一Protease 2AA site，我认为这
样只要细胞有gfp就一定有我的目标蛋白表达，
结果却不是我预测的，我在gfp表达细胞并没有监测到我的目标蛋白，
请问，各位做细胞系的大牛有何建议，谢谢 |
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s******y
发帖数: 28562 | 25 这个和lentiviral不一定有直接关系。我建议你先把那个载体直接表达在293T cell 里
面（不需要加包装载体），看看能不能表达出来两个蛋白。 |
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m**1
发帖数: 2 | 27 我想用Lentivirus介导针对某一基因ShRNA感染U2OS细胞以Knockdown该基因的表达，结
果是反复几次均未成功----感染U2OS细胞后用Puromycin筛选，细胞全部死亡。这说明
可能1）Lentivirus没有包装成功2）U2OS对Lentivirus敏感3）----？
我有该种Lentiviral表达质粒直接用Fugene
6转染U2OS细胞，然后用puromycin
筛选，有大部分细胞survival,说明Lentiviral表达质粒没有问题。
我包装Lentivirus的过程是：
Co-transfect下列质粒至 293T 细胞（用Fugene
6），48小时后，将上清液过滤，直接加到U2OS细胞上，48小时后加入Puromycin进行筛
选。
Lentiviral质粒 5ug
Gap 2ug
Rev 2ug
VSVG 2ug DNA总量 11ug, Fugene reagent 33ul.
第二次加大了DNA量，但还是不成功。
Lentiviral 质粒 10ug
Gap 3ug
Rev 3ug
VsVG 3ug DNA 总量19ug,Fugene 57 |
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d*******3
发帖数: 8598 | 28 J Gene Med. 2007 Jul;9(7):579-84.
Optimized production and concentration of lentiviral vectors containing
large inserts.
al Yacoub N, Romanowska M, Haritonova N, Foerster J.
Author information
Abstract
Generation of high titer lentiviral stocks and efficient virus concentration
are central to maximize the utility of lentiviral technology. Here we
evaluate published protocols for lentivirus production on a range of
transfer vectors differing in size (7.5-13.2 kb). We present a modified
virus prod... 阅读全帖 |
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g*********5
发帖数: 2533 | 29 What are lentiviral vectors?
Lentiviral vectors are a type of retrovirus that can infect both
dividing and nondividing cells because their preintegration complex (virus
“shell”) can get through the intact membrane of the nucleus of the target
cell. Lentiviruses can be used to provide highly effective gene therapy as
lentiviruses can change the expression of their target cell's gene for up to
six months. They can be used for nondividing or terminally differentiated
cells such as neurons, ma... 阅读全帖 |
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i*****i
发帖数: 154 | 30 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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s******o
发帖数: 187 | 31 I could not find the right lentiviral cloning vector that carries the SV40
large antigen. The ones on market are mostly in retrovirus.
I want a lentiviral vector to package SV40 lentivirus and then to
immortalize MEF cells.
anybody used lentiviral vector with SV40 large antigen? |
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g****0
发帖数: 425 | 32 Can I get the abstract as well?
How do you comment on this report?
Nucleic Acids Res. 2013 Mar 1;41(5):e63. doi: 10.1093/nar/gks1446. Epub 2012
Dec 28.
Differential integrity of TALE nuclease genes following adenoviral and
lentiviral vector gene transfer into human cells.
Holkers M, Maggio I, Liu J, Janssen JM, Miselli F, Mussolino C, Recchia A,
Cathomen T, Gonçalves MA.
Source
Department of Molecular Cell Biology, Leiden University Medical Center,
Eithovenweg 20, 2333 ZC Leiden, The Nether... 阅读全帖 |
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C**S
发帖数: 522 | 33 从一个网页上看到这段话：
For any lentiviral vector, the maximum insert size is 5.0kb, above which
will exceed the lentiviral virus packaging limit. This will result in
minimal or no packaging of viral particles. For complex lentiviral vectors,
such as dual promoter constructs or constructs with reporters and/or other
features, maximum insert size can be as small as 3.0kb. For insert sizes
over 5.0kb, removal of selection markers and selection marker promoters will
help to accommodate these larger inserts. |
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a***e
发帖数: 1010 | 34 Both siRNA and lentiviral shRNA methods are good enough to reach your
goal.
However, lentivirus usually has a higher infection efficacy than
transfection method. Therefore, using lentiviral shRNA may be a more
efficient
way to knockdown gene expression. |
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j**W
发帖数: 89 | 35 相关的文献实在不多。多谢！
CSH Protoc. 2008
Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version A.
Tiscornia G, Singer O, Verma IM.
CSH Protoc. 2008
Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version B.
Tiscornia G, Singer O, Verma IM. |
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l*******i
发帖数: 153 | 36 因为效率的问题，老板要求我试试miRNA cluster.前前后后试了三次，最初一周内
细胞生长加快，但始终没有colony出现；如果miRNA cluster和OSKM连用的话，倒是可
以非常显著地提高reprogramming的效率。
到目前为止，似乎只有两篇文章证实这个方法的，但效率差别太大；要命的是，第
一篇文章（号称效率提高至10%的那篇）是将miRNA302/367通过lentiviral vector整合
到genome上；而在moleculer therapy上的一篇文章表明，lentiviral vector本身就可
以将fibroblast诱导成iPS。第二篇文章通过连续四天转染mature miRNAs,可以得到iPS
，但是效率极为低（与第一篇文章用的miRNA有些不同）
我现在怀疑第一篇文章的可重复性：第一，单纯通过miRNA cluster，得不到iPS；
第二，即使能得到，效率应该也没有像该文章宣称的那样。联系文章作者，请教实验细
节，没有得到任何回复。不知版里有没有人试过这种方法？
现在越来越发现，iPS就是一个坑，临床应... 阅读全帖 |
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l***y
发帖数: 638 | 38 in my understanding, particle is virus, vector is backbone plasmid |
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d****i
发帖数: 2346 | 39 呵呵，也是从网上来的。。。。。。。。
One disadvantage associated with lentiviral vectors is the insert size. For
most lentiviral vectors developed, the maximum insert size is 5.0 kb. Insert
sizes less than 3.0kb can be efficiently produced at a high titer in
packaging 293T cells. Viral titers will be significantly decreased by
inserts longer than 3.0kb. As the SV40 genome is over 5.0kb, the expected
Lenti-SV40 titer is relatively low.
http://www.capitalbiosciences.com/system/data/files/15209699324 |
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f******g
发帖数: 1003 | 40 谢谢你的回复，我搜了一下，没有找到小鼠的sgRNA文库，只找到人的，
Human Lentiviral sgRNA Library - Cell Cycle (Plasmid #51046)
Human Lentiviral sgRNA Library - Nuclear (Plasmid #51047)
能耽误你几分钟的时间，贴的链接吗，谢谢 |
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h********r
发帖数: 519 | 42 Transcriptional responses in honey bee larvae infected with chalkbrood
fungus.
Aronstein KA, Murray KD, Saldivar E.
BMC Genomics. 2010 Jun 21;11:391.PMID: 20565973 [PubMed - indexed for
MEDLINE]Free PMC ArticleRelated citations
2.
Proteome profile and lentiviral transduction of cultured honey bee (Apis
mellifera L.) cells.
Chan MM, Choi SY, Chan QW, Li P, Guarna MM, Foster LJ.
Insect Mol Biol. 2010 Oct;19(5):653-8. doi: 10.1111/j.1365-2583.2010.01022.x
. Epub 2010 Jun 7.PMID: 20546039 [PubMed - ... 阅读全帖 |
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发帖数: 1 | 43 投入罕见病没那么容易，公司策略要很大调整。如果只做一两个没啥利润可图，做多了
会影响其他业务。除非弄个子公司，像sanofi 那样。Celgene应该还是集中在oncology
especially myeloma.
我对blue不是很看好,ex vivo成本太高，如果AAV能搞定且持续时间够长，margin比ex
vivo高太多，安全性也比较好。从更长远角度看，crispr干掉lentiviral vector是早
晚的事情。 |
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c******g
发帖数: 83 | 44 正在准备申请绿卡，想请大牛们帮忙给一些审稿机会。
弱背景简单在这里提一下
06土博毕业：遗传学 (熟悉基因克隆，表达，发育调控)
密歇根大学千老至今：
干过 microRNA调控心脏发育相关基因，
T-cell 相关的免疫疾病（GVHD and autoimmune disease）,
lentiviral vector-based,conditional gene expression system for transgenes or
shRNAs，
beta-globin disorders such as sickle cell disease and beta-thalassemia。
在此致谢大牛和过来人们！ |
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n******i
发帖数: 374 | 45 您好，希望能把这次机会给我，谢谢了
Yarong Liu
2623 Ellendale Pl.
Los Angeles, CA 90007
(213) 249-2419
y*******[email protected]
www.linkedin.com/pub/yarong-liu/2b/a36/65b/
PUBLICATIONS
1. Yarong Liu, April Tai, Kye-Il Joo and Pin Wang. “Visualization of DC-
SIGN-mediated entry pathway of engineered lentiviral vectors in target cells
” 2013, PLoS One 8(6): e67400.
2. Liang Xiao, Kye-Il Joo, Yarong Liu, Jinxu Fang and Pin Wang. “
Injectable thermo-sensitive hydrogel as an adjuvant: in vivo modulation of
dendritic ... 阅读全帖 |
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w****t
发帖数: 1 | 48
If things happened like that, why not design a control such as GFP insterted
into your vector, so it is easy to check the infection efficiency. It is not
surprised some cells survived after drug selection, there is why you need to
confirm the ectopic expression of your targer gene in stable cell line by WB
or other ways. |
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l**********n
发帖数: 240 | 49 如果用LENTIVIRAL VECTOR，情况就不一样了。 |
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n********k
发帖数: 2818 | 50 do u have the retro/lentiviral system with your system? was it linear or
circular? there are quite a few factors
need to be worked at before u can do it well if no one has done it before...
BTW, ur reasoning about the
transfection efficiency is a bit LOL:)) |
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