y*********u
发帖数: 183 | 1 Anyone here have experience how to add polyA signal when making knock in
allelle?
someone said that we should not add any polyA signal at all by which they
assume that they are using the orginal polyA signal of the knock in allelle | b*****o
发帖数: 150 | 2 You don't have to add polyA to your cDNA
The knockin construct normally has its own promoter and polyA region.The expression cassette will be randomly inserted into (any) genomic DNA.
What's your mean "allele"?
When use "allele" it normally means "the genomic region of the gene", you can delete, mutate or add (tags etc) to any part of your allele in vitro, then knock it in.
allelle
【在 y*********u 的大作中提到】
: Anyone here have experience how to add polyA signal when making knock in
: allelle?
: someone said that we should not add any polyA signal at all by which they
: assume that they are using the orginal polyA signal of the knock in allelle
| y*********u
发帖数: 183 | 3 Thank you for your reply!
Sorry for not making this clear. By saying knock in I am referring to the
method utilizing homology recombination to replace an original gene allele
by another one pre designed.
What you are saying is usually refereed as 'transgenic event'.
expression cassette will be randomly inserted into (any) genomic DNA.
can delete, mutate or add (tags etc) to any part of your allele in vitro,
then knock it in.
【在 b*****o 的大作中提到】
: You don't have to add polyA to your cDNA
: The knockin construct normally has its own promoter and polyA region.The expression cassette will be randomly inserted into (any) genomic DNA.
: What's your mean "allele"?
: When use "allele" it normally means "the genomic region of the gene", you can delete, mutate or add (tags etc) to any part of your allele in vitro, then knock it in.
:
: allelle
| g***y
发帖数: 201 | 4 1. A lot of expression vectors contain SV40 pA or BGH pA. You can simply
PCR adding those sequence right after the stop codon.
2. It all depends on how you design your KI allele. If your KI allele is
only to replace one exon, then you don't need to worry about anything. If
you are to insert a cDNA sequence right at the 1st exon,
then you may have to worry about non-sense mediated dacay. In this case, it
would be safer to add an exogenous pA.
allelle
【在 y*********u 的大作中提到】
: Anyone here have experience how to add polyA signal when making knock in
: allelle?
: someone said that we should not add any polyA signal at all by which they
: assume that they are using the orginal polyA signal of the knock in allelle
| y*********u
发帖数: 183 | 5 Thank you! what I did is completely replaced the exon1, exon2, and intron1
Do you think we need to add an exogenous pA signal in this case?
I added it anyway
it
【在 g***y 的大作中提到】
: 1. A lot of expression vectors contain SV40 pA or BGH pA. You can simply
: PCR adding those sequence right after the stop codon.
: 2. It all depends on how you design your KI allele. If your KI allele is
: only to replace one exon, then you don't need to worry about anything. If
: you are to insert a cDNA sequence right at the 1st exon,
: then you may have to worry about non-sense mediated dacay. In this case, it
: would be safer to add an exogenous pA.
:
: allelle
| y*********u
发帖数: 183 | | y*********u
发帖数: 183 | 7 We replaced the part of exon1,and exon2, do you think the endogenous pA
would work in this case? And could you post a link regarding the case of
utilizing the endogenous pA ?
it
【在 g***y 的大作中提到】
: 1. A lot of expression vectors contain SV40 pA or BGH pA. You can simply
: PCR adding those sequence right after the stop codon.
: 2. It all depends on how you design your KI allele. If your KI allele is
: only to replace one exon, then you don't need to worry about anything. If
: you are to insert a cDNA sequence right at the 1st exon,
: then you may have to worry about non-sense mediated dacay. In this case, it
: would be safer to add an exogenous pA.
:
: allelle
|
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