405 COMPARISON OF TARGETING PARAMETERS FOR THE HPRT GENE BETWEEN DOMESTIC AND OSSABAW SWINE

Abstract

Nuclear transfer using genetically engineered cells is the only method available to generate pigs with targeted genetic modifications. The Ossabaw swine has a unique genotype and can manifest components of the metabolic syndrome including insulin resistance, impaired glucose tolerance, and hypertension. Although the production of genetically modified Ossabaw pigs would be beneficial for modeling a number of cardiovascular and metabolic diseases, no work has been reported so far on gene targeting in Ossabaw pigs. We examined whether the targeting parameters obtained from domestic pigs can be used for gene targeting in the Ossabaw pig, using the hypoxanthine guanine phosphoribosyl transferase (HPRT) gene as a model. The HPRT gene is located on the X-chromosome and this makes it ideal for gene targeting studies in males. Fibroblast cells were isolated from 50-day-old Ossabaw fetuses. The cells were maintained in culture and exposed to 300 �g mL-1 of G418, an antibiotic to test the sensitivity of Ossabaw fibroblast cells to G418. The sensitivity was compared with that of fetal fibroblast cells isolated from domestic pigs. Sex of the Ossabaw cell lines was identified by amplifying a part of the SRY gene using primers designed for sexing in domestic pigs; DNA from adult male and female Ossabaw pigs was used as control. The CHORI-242 pig BAC library was screened for the HPRT gene using probes designed on the basis of the pig HPRT mRNA sequence from Genbank. Two fragments were subcloned and partially sequenced. Based on the sequences, primers were designed to amplify arms from Ossabaw male DNA for the targeting vector. Sexing of Ossabaw fetal fibroblast cells showed that 5 out of 8 isolated cell lines were male. After 5 days of G418 incubation, fibroblast cells of both domestic and Ossabaw pigs died due to exposure to the antibiotic. Primers designed on the basis of domestic pig HPRT sequences could successfully amplify 2 PCR products of 6 kb and 3.5 kb as the arms of the targeting vector. Sequence information of these fragments was compared to that of domestic pig DNA. A total length of 1657 bp was compared and we found a number of differences between the 2 types of DNA in both intron and repetitive sequences. The differences included 8 mismatches, 29 additional base pairs in the Ossabaw, and 2 extra base pairs in the domestic pig sequence (e.g. domestic pig DNA possessed a 27 bp microsatellite marker whereas the same marker in the Ossabaw pig was 52 bp long). Overall, Ossabaw fetal fibroblasts showed a sensitivity to G418 similar to that of domestic pig cells, indicating that neomycin resistance can be used as a positive selection marker for targeting Ossabaw pig genes. Although more sequence analysis is needed to draw a final conclusion, differences between Ossabaw and domestic pig HPRT sequences suggest that the targeting vectors produced from domestic pigs must be used with caution when targeting the gene in Ossabaw cells. This information for gene targeting in Ossabaw pigs will be useful for future studies in comparative medicine programs.