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generating cell line knockouts


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#1 ALW0012

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Posted 27 July 2010 - 04:14 PM

Question: Does anyone know if you can generate a cell line knockout in any cell line or does it have to be an embryonic stem cell? If so why?
I'm thinking about doing a series of experiments in HEK 293 cells and would love to KO my gene of interest to determine the effects on cell viability and was wondering if I can do that just by homologous recombination in HEK 293 cells. Any thoughts/ answers would be greatly appreciated.

#2 bob1

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Posted 27 July 2010 - 04:16 PM

You can definitely do KO in ordinary cell lines. There should be plenty of literature on the web that says how to do it.

#3 perneseblue

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Posted 27 July 2010 - 05:14 PM

You can definitely do KO in ordinary cell lines. There should be plenty of literature on the web that says how to do it.


You should also read up about the efficient of the cell line for homologous recombination. Some lines are more efficient, some are very very low.
May your PCR products be long, your protocols short and your boss on holiday

#4 Rsm

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Posted 27 July 2010 - 11:57 PM

One concern I'd have is the clonal power of your HEK cells. Most ES cells do have unlimited proliferative capacity, while HEKs don't. So you might need to do this several times before you get a good clone. You'll need to determine drug resistance before, HEKs are transformed with SV40 and Neo resistance, AFAIK. Other than that should be ok.

Cheers,

Minna
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