Dear Cristina,

Can you please clear that which vector you used for the transformation. Because Insertion site depends upon location of infection site, size of gene insert, type of vector and target site (in few cases). As you mentioned CAGGS promoter so along with that any reporter gene were used?

Regards

Piyush

Dear Cristina R

You can do TAIL-PCR or inverse PCR

You can use a reverse primer from the promoter backbone and 3 nested forward primers based on the organism genomic reseneces or unversal nested forward primers. Then do PCR and get sequenced the all PCR products. Based on the blast search in the mouse genome using query sequences of your product you can find out the positive result and also the insertsion site of your transgene.

Rest you can follow the TAIL-PCR or iPCR protocols.

Good Luck

Chet Ram

Thanks for your help we will try to do an inverse PCR following your recommendations.

@ Cristina,

Do you know the transgene copy number? If you have multiple insertions, it would be hard or complicated to use iPCR to figure it out. Check out the copy number of the transgene in your mouse.

Dear Yuan-Yeu. Yes I know that if we have more than one insert, iPCR will be unable to determine the insertion site. We are analyzing this now.

Thanks

Cristina

Hi Cristina,

If you have mice with multiple-copy transgene insertions, probably you can breed them with wild-type, and through genetic segregation to obtain mice with single transgene insertion (if the transgenes are not closely linked). Then you can use iPCR to determine the insertion location from the progeny. But, I know it will take much longer time.

Finally we requested the transgene sequencing service provided by Cergentis (www.cergentis.com). They successfully solved our query and provided us the information about transgene insertion using the TLA technology (at an affordable price). We fully recommend Cergentis services.