Transformation Of Human Growth Hormone (hgh) Gene F <sub> 4 </ Sub> Generation Of Red Carp Transit Plant Gene Location

Chromosomal in situ hybridization has been applied as a powerful tool to genelocalization. Based on that, PRINS and in situ PCR were developed for theidentification and localization to genes with fewer copies, even single copy. Whilethe integration and expression of heterogeneous genes in vivo has currently been inthe spotlight of the research to transgenic animal, the localization to heterogeneousgenes by chromosomal in situ hybridization would supplies the primary informationon the mechanism of integration, targeting integration, and the relation ofintegration and expression of heterogeneous genes. In this study, we attempted tolocate the heterogeneous genes in the chromosome set of transonic red carp F4offspring by in situ hybridization, PRINS, and in situ PCR, respectively, to whichthe DIG-labeled alkaline phosphatase system was applied.With the DIG-labeled hGH as probes, we performed the chromosomal in situhybridization to the metaphase chromosomes in whole-blood cultured lymphocyteof the transgenic red carp F4 offspring. The result showed that the heterogeneousgenes distributed over the chromosome set with the pattern of the multiple sites, themajority of which appeared in region of near- centromere or telomere, and few inarm of chromosomes. No specific pattern of distribution exhibited.Applying the method of in situ PCR, we amplified the hGH gene for 25 roundsin slides of prepared chromosome with a pair of primers the same as that in PRINS,while the identical dyeing processing was applied. Having compared the result withthat of in situ hybridization and that of PRINS, we could observe the apparentsignals from heterogeneous genes in region of near- centromere or telomere, even inarms of chromosomes. Therefore, we could conclude that the method of in situ PCRis the best for the identification of genes with single copy or low numbers of copiesamong all three methods mentioned above.Summarizing the results of identification and localization to heterogeneousgenes by the three methods mentioned above, we concluded as follows: theheterogeneous genes had been integrated into the genome and could be checked upin most of the chromosomes during the cleavage phase; there was generally one sitein an identical chromosome for integration; no specific pattern was revealed for theintegration of heterogeneous genes in the chromosome sets. To be analyzed, theintegration of heterogeneous genes may be subject to the mechanism we called" limited random integration".