发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: A question about KO
发信站: The unknown SPACE (Fri May 30 23:04:46 2003), 站内信件

windysea, it seems that you have to do KO job as well. good:).
typical design for the targeting construct will aim at the
first several exons. in my case, since i only got the genomic
fragment in the middle, and several years ago mouse genome
sequence was not published yet, i had to choose a functional
domain that is important to the gene, in my case, a protein
kinase. when i got the knockouts, the phenotype was pretty
interesting so that i could get some publications; however,
i still could not get a clean western blot to show the absence
of the protein, particularly the N-terminus. as i sequenced
the mutant RNA, i found the splicing events were intact, except
skipping of the deleted exons. so it means that the truncated
mutant RNA is transcribed and present in considerable amount,
yet not as my boss said that typically mutant RNA would be
quite unstable. so when you started the isolation of the
genomic, you MUST be aware of a couple of things:
single gene? redundant pathway? worth doing?
genetic background? 129 ES cells have higher chance for germline
transmission while transplanted into C57 blastocyst, and you
MUST make sure that the transgenic facility at yours have
successful experience and make sure of the genetic background
of the ES cells (129 has many substrains). i used 129 SvEvTac,
it gave me good result. so you have to isolated the clone from
a library or synthesize it from genome with exactly the same
genetic background.
existence of other genes? orientation? which exon to KO?
假如上天能再给我一次机会,我会这样
add some marker such as GFP (may be toxic?) or IRES-lacZ, or
even loxP/lox511 (refer to the Nat Biotech paper) to monitor
the recombination ...
in the case of Cre/loxP system, setup mating pairs of strains
with floxed gene with EIIa-Cre to speed up generation of
heterozygote; meanwhile, if germ-line transmission succeed,
setup mating pairs to generate congenic mice from the very
beginning ...
【 在 windysea (神仙眷侣~我怀念你爱上我的第一天) 的大作中提到: 】
: when you design KO by deleting one or two key exons in the middle,
: how do you know in advance it will result in a truncated protein which is not
: functional, or no protein?


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※ 修改:．Marble 于 Jun 2 15:06:43 修改本文．[FROM: 128.118.]
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.118.]