w***e
发帖数: 269 | 1 Here is the problem. We recently developed a mouse knockout line with a
company. The entire gene is knocked out with all the exons and introns
being replaced with a reporter gene (lacZ) cassette. We got the
heterozygous (+/-) mice and I did the breeding. But now I have trouble
genotyping to find out the knockout mice (-/-). I tried two pairs of
primers in that gene to PCR out ~400 bp fragments in the last exon (the
biggest exon which codes the functional domain of the protein). PCR with
one pair of primers didn't give any bands. The other PCR seemed worked
with a faint band at the predicted size for most of mice and no band for
about 1/4 of them (5 out of 16, which i assume is the correct Mendilian
ratio for the knockout). But because the bands were faint and there were
many non-specific bands, it didn't look very convincing to me. BTW, I did
use the touch-down PCR stategy to reduce non-specific amplification.
Because we are working on a brand new protein, there is no good antibody
against it. What are my options to confirm which ones are knockout mice? I
assume I could kill one or two and do RT-PCR to look at the mRNA or design
another pair of primers and hope it might give a clear-cut result. But if
there are other better options, I would love to know. PCR genotyping for
the reporter gene works fine. We just need a way to tell -/- from -/+,
both of which have the engineered reporter cassette. Thanks a lot! | w*e
发帖数: 740 | 2 我感觉用比较好的LACZ 引物就应该可以/
看看哪个文献上报道其他LACZ的KI老鼠，如果他们的引物能同时PCR出WT 和LACZ
不同的2个带，你就应该可以用（如果你的老鼠本身造的没问题）。
具体设计引物，估计你得咨询做TARGET VECTOR的哪个人
【在 w***e 的大作中提到】
: Here is the problem. We recently developed a mouse knockout line with a
: company. The entire gene is knocked out with all the exons and introns
: being replaced with a reporter gene (lacZ) cassette. We got the
: heterozygous (+/-) mice and I did the breeding. But now I have trouble
: genotyping to find out the knockout mice (-/-). I tried two pairs of
: primers in that gene to PCR out ~400 bp fragments in the last exon (the
: biggest exon which codes the functional domain of the protein). PCR with
: one pair of primers didn't give any bands. The other PCR seemed worked
: with a faint band at the predicted size for most of mice and no band for
: about 1/4 of them (5 out of 16, which i assume is the correct Mendilian
| w*e
发帖数: 740 | 3 还有验证 GENOTYPING根本不需要 RT-PCR
【在 w***e 的大作中提到】
: Here is the problem. We recently developed a mouse knockout line with a
: company. The entire gene is knocked out with all the exons and introns
: being replaced with a reporter gene (lacZ) cassette. We got the
: heterozygous (+/-) mice and I did the breeding. But now I have trouble
: genotyping to find out the knockout mice (-/-). I tried two pairs of
: primers in that gene to PCR out ~400 bp fragments in the last exon (the
: biggest exon which codes the functional domain of the protein). PCR with
: one pair of primers didn't give any bands. The other PCR seemed worked
: with a faint band at the predicted size for most of mice and no band for
: about 1/4 of them (5 out of 16, which i assume is the correct Mendilian
|
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