Identification Of Transgene Integration Loci In Human Alpha-lactalbumin Cloned Transgenic Cattle

In recent years, the production efficiency of transgenic animals has been greatlyimproved through the combination of transfection and somatic cell nuclear transfer(SCNT), but the process (mechanisms) of chromosomal integration of transgenes is stillnot fully understood. Thus we examined the transgene integration sites by first cloning theflanking sequences of the exogenous gene that expresses human alpha-lactalbuminproduced by SCNT obtained from five transgenic cattle by using TAIL-PCR and JunctionPCR, in order to get on the basis of the process (mechanisms) of chromosomal integrationof transgenes.The results were as follows:1. Twenty-six3’ flanking sequences were obtained by using the thermal asymmetricinterlaced PCR (TAIL-PCR) and divided into three types (type A, B, C) according to theircharacteristics of the five transgenic cattle which expresses human alpha-lactalbuminproduced by SCNT.2. A commonly observed phenomenon in3’ flanking sequences was that the transgeneall broke at the same nucleotide site15821and connected with an unknown sequence(UN1) about300bp length, which has a227bp consensus part. Another unknownsequence (UN2) was found in type B and C.3. We failed to obtain the5’ flanking sequences by using TAIL-PCR, but three5’border sequences were cloned by using specific-primer PCR. We obtained three5’ flankingsequences respectively belong to081223,172,189, constituting three complete integrationsites. The consensus sequence of the topoisomerase I cutting (cleavage) sites was found inthe chromosomal DNA of5’ flanking sequences and exogenous gene of3’ flankingsequences.4. In sample081223, the transgene was inserted in chromosome1of the host genome.The3’OH boundary is flanked by position15821of the transgene with a3875bp deletion.And there is an262bp length unplaced homologous fragment (UN1) is connected to5’chromosome sequence. A short fragment46bp length deletion in the5’ border sequence ofthe transgene.5. In sample172, the transgene was inserted in chromosome2of the host genome. The3’OH boundary is flanked by position15821of the transgene with a3875bp deletion.And there is an263bp length unplaced homologous fragment (UN1) is connected to5’chromosome sequence. A short fragment76bp length deletion in the5’ border sequence ofthe transgene.6. In sample189, the transgene was inserted in chromosome24of the host genome.The3’OH boundary is flanked by position15821of the transgene with a3875bp deletion.And there is an266bp length unplaced homologous fragment (UN1) is connected to5’chromosome sequence. A short fragment98bp length deletion in the5’ border sequence ofthe transgene.According to our results, indeed, there are some common characteristics and complexrearrangements that exist in the insertion sites of cloned transgenic cattle, which provide animportant basis for the research of transgene genetics.