h*****y
发帖数: 10 | 1 从公司订制的KO老鼠,带CRE基因,大半年折腾下来去掉CRE并拿到大量杂合子,按照公司
给的DATA SHEET, GENOTYPING双引物鉴定老鼠各基因表型正常,符合孟德尔遗传,但在对
培养的野生型和纯合子MEF细胞鉴定时,我发现虽然公司给的GENOTYPING鉴定没问题,但
我用RT-PCR和WB鉴定KO纯合子细胞时却都能发现该基因蛋白表达(重复多次),所以现在
感到非常困惑,我鉴定做WB的抗体是找公司制备的,非特异条带虽然有点多,但目的大小
附近有条带,而REAL-TIME PCR我之前一直用来鉴定SIRNA的KNOCKDOWN,效果也非常好,难
道是出现了假KO的情况? | h*****y
发帖数: 10 | | H****N
发帖数: 997 | 3 How was the KO made, by homologous recombination or gene trapping? Why is
there cre in it? If the mouse is made by gene trapping, it is possible the
gene is not knocked out completely, depending on where the gene trapping
cassette is located. Another remote possiblity is that there is another
related or duplicated gene in the genome, and thus your results. | O******e
发帖数: 4845 | 4 刚开始拿到老鼠的时候，你们做soutern blot了嘛？
【在 h*****y 的大作中提到】
: 从公司订制的KO老鼠,带CRE基因,大半年折腾下来去掉CRE并拿到大量杂合子,按照公司
: 给的DATA SHEET, GENOTYPING双引物鉴定老鼠各基因表型正常,符合孟德尔遗传,但在对
: 培养的野生型和纯合子MEF细胞鉴定时,我发现虽然公司给的GENOTYPING鉴定没问题,但
: 我用RT-PCR和WB鉴定KO纯合子细胞时却都能发现该基因蛋白表达(重复多次),所以现在
: 感到非常困惑,我鉴定做WB的抗体是找公司制备的,非特异条带虽然有点多,但目的大小
: 附近有条带,而REAL-TIME PCR我之前一直用来鉴定SIRNA的KNOCKDOWN,效果也非常好,难
: 道是出现了假KO的情况?
| h*****y
发帖数: 10 | 5 It's a homologous recombination deleting one exon. And this gene is a member
of protein family.
【在 H****N 的大作中提到】
: How was the KO made, by homologous recombination or gene trapping? Why is
: there cre in it? If the mouse is made by gene trapping, it is possible the
: gene is not knocked out completely, depending on where the gene trapping
: cassette is located. Another remote possiblity is that there is another
: related or duplicated gene in the genome, and thus your results.
| h*****y
发帖数: 10 | 6 没做过，老大。 不过公司的GENOTYPING 引物有两对，一对设计在敲掉的外显子两侧，
另一个设计在敲掉的外显子上，感觉这两对引物PCR检测外显子有没有被KO掉，还是蛮
可靠的。
【在 O******e 的大作中提到】
: 刚开始拿到老鼠的时候，你们做soutern blot了嘛？
| D*a
发帖数: 6830 | 7 既然是cre来ko的，是不是cre的表达量和表达的cell type问题？ | D*a
发帖数: 6830 | 8 你的意思是两个loxp外侧还是内侧？
一对设计在敲掉的外显子两侧，
【在 h*****y 的大作中提到】
: 没做过，老大。 不过公司的GENOTYPING 引物有两对，一对设计在敲掉的外显子两侧，
: 另一个设计在敲掉的外显子上，感觉这两对引物PCR检测外显子有没有被KO掉，还是蛮
: 可靠的。
| h*****y
发帖数: 10 | 9 CRE是公司用的ES细胞转基因带来的为了方便重组,我们拿到老鼠后通过杂交已经把CRE
去掉了,现在我们的老鼠里已经没有CRE了.
【在 D*a 的大作中提到】
: 既然是cre来ko的，是不是cre的表达量和表达的cell type问题？
| h*****y
发帖数: 10 | 10 第一对引物设计在LOXP外侧,第二对引物中一个引物在外侧另一个在里面
【在 D*a 的大作中提到】
: 你的意思是两个loxp外侧还是内侧？
:
: 一对设计在敲掉的外显子两侧，
| | | D*a
发帖数: 6830 | 11 对cre 很好奇，能告诉是什么cre和怎么作用不？ | H****N
发帖数: 997 | 12 It seems to me that your western is not reliable. As to the RT-PCR, you
should use primers spaning the dedleted exon to check that exon is actually
gone. Remember, after deleting one exon, the rest of the gene can still be
transcribed and make a deleted mRNA.
member
【在 h*****y 的大作中提到】
: It's a homologous recombination deleting one exon. And this gene is a member
: of protein family.
| D***a
发帖数: 516 | 13 那这个 deleted mRNA 能翻译成蛋白吗？
actually
【在 H****N 的大作中提到】
: It seems to me that your western is not reliable. As to the RT-PCR, you
: should use primers spaning the dedleted exon to check that exon is actually
: gone. Remember, after deleting one exon, the rest of the gene can still be
: transcribed and make a deleted mRNA.
:
: member
| O******e
发帖数: 4845 | 14 Is this possible that your targeting cassette was inserted somewhere else?
Make sure the primers you were using can tell you if they are amplifying
your gene locus, not the targeting cassette.
One round of Southern blot should answer all your questions.
【在 h*****y 的大作中提到】
: 没做过，老大。 不过公司的GENOTYPING 引物有两对，一对设计在敲掉的外显子两侧，
: 另一个设计在敲掉的外显子上，感觉这两对引物PCR检测外显子有没有被KO掉，还是蛮
: 可靠的。
| H****N
发帖数: 997 | 15 It depends. If the rest of the mRNA is still in frame, a truncated protein
can still be produced. Remember that lz does not have definitive evidence
that a protein can still be produced from the ko allele.
【在 D***a 的大作中提到】
: 那这个 deleted mRNA 能翻译成蛋白吗？
:
: actually
| D*a
发帖数: 6830 | 16 还有要看你们当时是怎么决定的KO的strategy吧
像我们的老鼠敲掉的是跨膜蛋白的ligand结合位点的exon，然后敲掉之后在下一个exon
中造成移码突变，然后造成几个stop，这样就确保蛋白质没有作用。
还有我们的引物设计是这样的
引物a －－loxp－exon－－引物b－－loxp－－反向引物c
这样wt和lox是靠bc来区别，ac相比之下太长扩增效率很低，ko之后就是ac扩增，这样
wt lox excised都可以用这三个引物来区分。我觉得这种pcr比你的好些，要不你自己
重新设计引物试试吧。 | a*****g
发帖数: 543 | 17 what kind of Cre did you use?
If you happened to have SOX2-Cre, you will be able to get good KO( of target
ed site).
I'm assuming you are doing appropriate control of the genotyping, with both
WT primers and recombinant primers...
How big was the targeted exon? where is your qRT-PCR targeting? Having one p
rimer locating in the exon may help to clarify whether deletion of the exon
occurred...
【在 h*****y 的大作中提到】
: 从公司订制的KO老鼠,带CRE基因,大半年折腾下来去掉CRE并拿到大量杂合子,按照公司
: 给的DATA SHEET, GENOTYPING双引物鉴定老鼠各基因表型正常,符合孟德尔遗传,但在对
: 培养的野生型和纯合子MEF细胞鉴定时,我发现虽然公司给的GENOTYPING鉴定没问题,但
: 我用RT-PCR和WB鉴定KO纯合子细胞时却都能发现该基因蛋白表达(重复多次),所以现在
: 感到非常困惑,我鉴定做WB的抗体是找公司制备的,非特异条带虽然有点多,但目的大小
: 附近有条带,而REAL-TIME PCR我之前一直用来鉴定SIRNA的KNOCKDOWN,效果也非常好,难
: 道是出现了假KO的情况?
| h*****y
发帖数: 10 | 18 Thanks everybody for many useful suggestion!
I guess maybe it's my identifying problem because my ab is not good and my Q
-PCR primers are also just targeting another exon. I will try to design new
identifying primers tageting the deleted exon. |
|
