A premade stable lentiviral packaging cell line including expression vector.

[x11...@mail.com]

Alright this is a shot in the dark but if anyone can understand what I'm talking about and knows a company that sells custom stable lentiviral packaging cell lines (293t cells) which have already been transfected with some expression vector of a customers choosing please let me know. I know there are some that sell cell lines with lentiviral packaging vectors but can't find any that will transfect an expression vector in for me. The problem is that it's sorta easy to transfect cell lines will an expression vector and lentiviral packaging plasmids but this only results in transient lentiviral particle expression, that is to say the cells stop producing lentivirus particles after a few days. It is possible to make stable packaging cell lines it's just really hard (I can't do it on my own). I need a cell line that will produce lentivirus particles (which then can be used to integrate a gene of my choosing in other cells) for a long time (several months at least). So if anyone knows a company that sells or will make such a cell line please post a response.

[Josiah Zayner]

What are you trying to do?

Why not just make stable cell line?

[x11...@mail.com]

I think I may have misunderstood something. If I transfect some premade stable packaging cell line with some expression vector will it stably produce lentiviral particles which will integrate whatever DNA is on the expression vector or will it only transiently express such particles and then revert back to producing only the default proteins of the packaging cell line. For example lets say I have the stable cell line which is expressing varius proteins needed for lentiviral particle assembly (ie the envelope, integrase, etc) I then transfect it with an expression vector for GFP. Now it should for at least a few days produce lentiviral particles which will integrate GFP into whatever cells are transduced with it. But will the packaging cell line continue producing such lentiviral particles (ie containing capable of integrating GFP) or will the cell line eventually stop expressing the information on the expression vector?

[Nathan McCorkle]

Have you simply googled lentiviral expression packaging kit? I see lots of hits.

I'm also going to go ahead and say for others reading this, if not yourself also, that using a product like this is definitely unsafe in less than a BSL-2 (bio safety level) lab. I would not recommend this project for the average DIYbio person.

http://www.safety.duke.edu/SafetyManuals/Lab/Section1BiologicalSafety/Chapt%208%20BIOSAFETY%20LEVELS.pdf

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[Cathal Garvey]

You probably know this already if you know enough to ask that question, but..
..be careful with lentiviral vectors, or any other vector capable of transforming mammalian cell lines in-vitro. Because they can still efficiently transduce those genes into cells in-vivo, for example when you breathe the aerosol produced by most micro-pipette tips while handling viable lentivirus particles.

Having studied viral/nonviral gene therapy techniques for years, I can comfortably tell you that most lentiviral vectors are NOT designed for safe use, and those that are alleged to be "safe" usually aren't. Lentiviruses, and the vectors that derive from them, tend to inject their DNA semirandomly into genomic regions, usually near to gene-rich regions, and if they carry a strong promoter (usually the case, especially for whiz-bang GFP projects), they can and do happily activate oncogenes downstream. At the very least, they can cause nearby heterochromatin to unwind, leading to similar activation of unwanted genes.

In all likelihood, such mutant cells will get killed by the immune system in short order (MHC presentation of GFP tends to raise T-cell eyebrows); I imagine this is why cases of experimenters with self-inflicted cancer are not very common. But it should be considered when working with lentivirii, adenovirii or even adeno-associated virii: they can transfect YOU as well as the cell line. At least with AAV, genomic integration is usually pretty predictable..

www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey

[Mega [Andreas Stuermer]]

http://emboj.embopress.org/content/32/19/2645

came across this. Promoters can affect histone density. That was what you mentioned, Cathal.

Btw, I found out histones are "basic" / alkaline proteins.