The Production Of Gene-Targeting At GEF β-Casein Gene Locus With Human Lactoferrin Gene And Analysis Of Gene Integrate Site

[Objective]In order to select appropriate transgenic cell clones which can be used as donor cells to produce cloned animal by nuclear transfer, we set up GEF clones which gene-targeting with hLF Gene in P -casein Gene Locus , then examined the viability , chromosome ploidy and the integration site of hLF Gene. [Methods]1. After transfection of GEFs with nBC-LF gene, the cells were incubated in G418 and GANG containing medium and further analyzed by PCR and Southern blot for targeting events.2. Analysed the karyotype of the normal GEFs and transgenic GEFs . During the selection under G418 and GANG containing medium, the viability of transgenic GEFs was examined.3. FISH technique was used to detect the targeted gene and determine whetherthe homologous recombination occurs. The β -casein gene probe and hLF gene probe were hybridized with chromosomes of transgenic GEF and normal control GEF each other. [Result]1. We have gotten six GEF clones that gene-targeting with hLF Gene in β -casein Gene Locus and analyzed the clones by PCR and Southern blot.2. The chromosome number of normal GEFs was normal in 20 generation . Afer the 20th generation the viability of the cells was reducing and ploidy was changed differently. When chromosome counting of cells was performed, it was found that 2 clones from 4 clones have normal chromosome number, with more than 80% diploid cells. The karyotype analysis of 9# clone was identical with the standardkaryotype of goat, so the 9# clone can be used as donor cells to produce cloned animal by nuclear transfer.3. FISH signals showed that P -casein gene and hLF gene locate in the same position of No. 6 chromosome and the homologous recombination occured. [Conclusion]One of the character of making transgenic animals by somatic cell-mediated is that the transgenic processing focus on cell level, so setting up the appropriate transgenic cell clones which can be used as donor cells to produce cloned animal is the most important part. PCR/Southern blot/FISH system can investigate the integration site of foreign gene in transgenic cells exactly and determine if the homologous recombination occur. The karyotype analysis can select the clone with normal chromosome. These work offer the important experimental foundation for the technique route of making transgenic animals by somatic cell mediated.