The targeting vector we used to produce our perforin knockout mice contains a 1.2 kb piece of a Neo-resistance cassette inserted into the SmaI site of exon 2. We have recently generated some oligos for the purpose of testing tail DNA for the perforin genotype using PCR. P17 is a primer that hybridizes to sequences in intron 2, and P12 is a primer that sits just 3' of the SmaI site in exon 2. Wildtype DNA gives a band of about 500 bp. Because of the Neo insertion between the two primer sites when the gene has been disrupted, perforin knockout DNA gives a band of about 1600 bp. Since 1600 bp is not always efficiently amplified, you may use P26 (which sits inside the Neo cassette) to detect knockout DNA. Knockout DNA will produce a band of about 350 bp (and the faint 1600 bp band). Heterozygotes should have a 350 bp (ko), 500 bp (wt) and a faint 1600 bp (ko) band. Homozygous wildtype mice will have only the 500 bp band.

P17: 5' - CGT GAG AGG TCA GCA TCC TTC - 3'

P12: 5' - TGG CCT AGG GTT CAC ATC CAG - 3'

P26: 5' - ATA TTG GCT GCA GGG TCG CTC - 3'

For cycling conditions, we use 1 µl of DNA from our tail-preps (usually, DNA recovered from 1 cm tail is resuspended in 200 µl, so this is 1/200th of a piece of tail...well, you get the point. I'd guess this is about .5 to 1 µg of DNA). Also, we use 1.5 mM MgCl2, if it really makes a difference. Cycle is:

Temp Time

94oC 1.5 min

57oC 2.0 min

72oC 5.0 min

We repeat the cycle 30-35 times, and then run about 1/5 of the reaction contents on a 1.2% agarose gel with ethidium bromide. It usually works fine, but there are occasional samples that don't seem to amplify. For these, we generally do a phenol/chloroform on the bulk DNA, and then they seem to amplify appropriately.

Good luck and let us know if you need additional information.

Craig Walsh, Ph.D.
February 1995