Dear Frauke,
It depends on the distance between the recombination breakpoint and your GFP.  We put a point mutation into the 5' end of histone H3a, with the selectable marker about 600bp downstream.  About 50% of targeted alleles had the substitution.  This may very from gene to gene.  You will have to sequence a PCR product from the endogenous locus to make sure your recombinant doesn't have the bad sequence.  Obviously it's good to have a gene that targets well.
Jonathan


Dr. Jonathan Chubb
Division of Cell and Developmental Biology
School of Life Sciences
University of Dundee
Dow Street
Dundee
DD1 5EH
+44 (0) 1382 386336
>>> Frauke Bach  08/18/11 4:39 PM >>>
Dear Dicty community,

We are planing to knock in a gfp tag downstream a gene of interest. Therefore the upstream flank of the construct lies in the coding region of the gene. To generate the knock-in construct we had to introduce a restriction site at the 5'end of the upstream flank.
Our question is, if the few additional basepairs of the restriction site will also be integrated into the genome (even though, they are not part of the homologous region). In our case this would lead to a frameshift within our target gene which would be detrimental.
Are there easy ways to get around this problem (if no endogenous restriction site is present)?
  
Greetings from Hamburg,
Frauke


Bernhard-Nocht-Institute for Tropical Medicine
Research Group Hagedorn
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