Question

6a) You want to use homologous recombination to generate a mouse that does not express a functional XPC gene (‘Knock-out’ or KO). To do this, you want to delete exon 10 and replace it with a gene that confers resistance to the drug Neomycin (NEO. The usual second selection with tk we discussed in class is not shown here). Using the diagram as a guide: illustrate on the diagram where you expect crossing over to occur and (below the diagram) draw the expected genomic locus after integration. Include the EcoR1 digestion sites.

You perform your experiment and integrate your targeting construct into the mouse genome in embryonic stem cells (ie you get neomycin resistant cells). Before injection into blastocysts to make transgenic mice, you want to confirm that the integration worked properly: you digest genomic DNA with the restriction enzyme EcoRI and perform a Southern blot using the probe indicated in the diagram. You get the following results (remember the integration may be heterozygous at this point, also remember that the probe will only pick up the fragment that contains the region of the target that matches its sequence).

6b) Which band is the normal the normal allele? Why?

EcoRI ECORI exon 10 8 9 11 12 Southern probe EcoRI NEO 11 A - B

Answer 1

6a) crossing over occurs in the regions of homology here homologous regions in these 2 strands of DNA are exons 9 and 11 so crossing over happens in those regions and in the final construct after recombination will not have exon 10 instead of that NEO gene will be present there. 6b) recombined DNA has 3 EcoRI site, so when digest with EcoRI recombined DNA gives smaller fragment compared to normal gene so it will migrate faster than normal allele ( probe is in the exon 8 so it will recognize both bands) so the lower band corresponds to the recombined band.

NEO i 2 A Zk