Research Of Exogenous Gene Copy Number And Integration Site In Transgenic Cell Lines And Animal

In the process of somatic cell nuclear transfer technology to produce the transgenic animals, exogenous DNA will be inserted into the genome any position randomly when into the recipient genome. Inserted gene may be disrupted the transcription process of host cell gene, or activate harmful genes, leading to the fetal malformation or death, resulting transgenic animal survival rate was low. In addition, the phenomenon of exogenous gene silent in transgenic animals was often happens, in which was the exogenous gene in host chromosomal location due to gene silent, the integrated sites of exogenous gene has a crucial effect on the expression levels of exogenous gene. Thus, the cell lines of identificated positive was can not for nuclear transfer necessarily. Through to detected the exogenous gene of copy and integration sites in positive cell lines, to research the integration mechanism of foreign gene. It can help us analyze the impact of exogenous gene for future transgenic animal’s genetic traits. Cell lines could be used in somatic nucleus transplant experiment and avoid blindness production of transgenic animals.Meanwhile, in the current,the production transgenic animals of the has clear genetic background is the requirement and the inevitable trend of the transgenic animal’s industrial development.Myostatin（MSTN） is a negative regulator of skeletal muscle development and growth, bovine MSTN gene by controlling myoblast size, number and proliferation rate. MSTN gene specific expresses in muscle tissue. Lower and loss of MSTN activity will lead to excessive development of animal muscle, which shows the diameter or the number of muscle fibers become larger or more. MSTN interference vector （pGenesil-1.1-MSTN） can promote the muscle growth.In this study, we used MSTN interference vector to transfect Luxi cattle fetal fibroblasts and obtained transgenic cell lines. Using real time PCR and high-efficiency thermal asymmetric interlaced PCR, we detected expressing copy of MSTN plasmid and their integration sites in bovine genome, analyzed the characteristics of the integrate exogenous genes in transfected cell lines. To selected the cell lines suitable for somatic cell nuclear transfer technology to produce the transgenic animals, to avoid the blindness in the production of transgenic animals.To explore the mechanism of Integrate of exogenous genes, we detected the exogenous gene copy number and integration sites of transgenic cattle. The main research results are as follows:1.To explore the optimal suitable bovine fibroblast cells transfected with the conditions:gene transfection into Luxi cattle fetal fibroblasts by liposome mediated, cells were transfected in6 well plates, the optimum amount of plasmid was2.5μg per well, the ratio of quality of the plasmid and transfection reagent was1:4would get the best transfection efficiency.2.Obtained the positive transgenic cell lines of MSTN interference vector to transfect bovine fibroblasts, we detected expressing copy of MSTN plasmid and their integration sites in bovine genome. The results showed that the copy numbers of plasmid were effectively detected by quantitative PCR in the five positive cell clones which were2.26±0.32,1.52±0.25,25.68±1.02.8.43±0.73and6.72±0.1respectively.3.Using hiTAIL-PCR method to obtained two binding fragments of exogenous gene integration site. A fragment of the foreign genes was integrated in the bovine chromosome16genomic clone （NW₀03104426.1）. B fragment of the exogenous gene into bovine chromosome21, located on the CHRNA7genes. We explored the mechanism of integrated of exogenous genes through compared the integration site sequence features. Integration sites detected by hiTAIL-PCR showed that there was recombination in the process of insertion of plasmid fragment into target genome. And there was a short homologous sequence in the junction of exogenous genes and chromosomes.4.The copy numbers of plasmid were effectively detected by quantitative PCR in the transgenic cattle which were1.9±0.11、3.6±0.57and3.7±0.13respectively. Using hiTAIL-PCR method to detecting the integration site transgenic cattle. The results showed thatexogenous gene into the chromosome2, the insertion site between ACTR3and DB1. Foreign genes was recombination in the process of insertion into target genome.This experiment using real time PCR and high-efficiency thermal asymmetric interlaced PCR, we detected copy numbers of foreign genes and their integration sites in bovine genome. We explored the mechanism of integrated of exogenous genes. It provided important theoretical and experimental foundations for the production of transgenic cow.