Integration Site Analysis Of The Exogenous Gene In Human Lysozyme Cloned Transgenic Cattle

Recent progress in animal cloning has provided an attractive alternative to improvetransgenic efficiency, through the combination of transfection and somatic cell nucleartransfer （SCNT）. Transgenic animals have been used to produce important proteins,livestock bioreactor, study copy number, integration site, cultivate the disease-resistant strains,xenotransplantation donor and production of bio-pharmaceutical products. It has shown a broadapplication prospects.To determine the integration sites of transgene is the primary and important task afterobtained the cloned transgenic animals. Therefore, in our study, we successfullydetermined the integration sites of transgene in cloned transgenic cattle, with in-depthanalysis of the chracteristics of the integration sites.The results were as follows:1. The whole integration sites of two samples and the3’ flanking sequence of onecattle were successfully cloned by the combination of TAIL-PCR and Junction PCR.2. In sample0503, the exogenous genes was integrated into the genomic clone（NW₀03104315.1） in chromosome12. The integration of exogenous gene caused notonly3875bp deletion of3’ border and314bp deletion of5’ border of the exogenous gene,but also a57bp deletion of chromosome DNA. Between the3’ border sequence and thechromosome DNA, there was a290bp unknown fragement. The5’ border sequence ofsample0503were closely connected with the estriction enzyme cutting sites oftopoisomerase I （5’-A/T-G,C-T/A-T/A-3’）.3. In sample Fuyang, the exogenous genes was integrated into the genomic clone（NW₀03104497.1） in chromosome19. The integration of exogenous gene caused notonly3875bp deletion of3’ border and104bp intemitent deletion of5’ border of theexogenous gene, but also a94bp deletion of chromosome DNA. Between the3’ bordersequence and the chromosome DNA, there was an unknown fragement30bp shorter thanthat of0503.227bp of this fragment was exactly the same as that in0503. The5’ bordersequence of sample Fuyang was also closely connected with the estriction enzyme cuttingsites of topoisomerase I.4. The primers for the verification of3’ flanking sequence of the exogenous gene were used for the amplification of sample1261, and part of the3’ flanking sequence wassuccessfully obtained. The result showed that, the3’ border of sample1261was broken inthe same position as that in0503and Fuyang. The227bp unknown fragment which wassame as that in the other two samples was closely connected with the exogenous gene. Dueto the ralevant chromosome information hasn’t been obtained, we could not determin theexact position of the transgene in host genome.