| Object: To study ET clone technology and its application, as well as the function of secreted gene Colec11, whose function is remain unknown. We need to construct a gene-targetting vector of mice. In human genome, Resp18 is a secreted protein gene, which function is unknown yet. This gene has the structure of signal peptide at the N'terminal, without transmembrane domain. Our study aimed to investegate the function of this gene by obtain the Resp18 gene knockout mice. Thereby evaluate the possibility of developping a new medicine by the gene, and establish foundation for searching drug screening sites. The general object of this study is to develop the DNA engineering platform—ET recombineering and establish Resp18 gene knockout mice model, and provide fundation for the futhur investigation of the function of Resp18 gene and the possibility of development of new drugs.Methods: First, we obtain the large fragment genomic DNA from BAC DNA by PCR amplification, then replaced the area of knockout by selection marker--neo. Finally we got the gene targetting vector of Colec11. And in this study, we get the detailed information of sequence,transcription and protein of Resp18 gene in mouse genomic by bioinfomatics, and obtain the number of BAC clone which contains whole gene of Resp18. By the application of ET recombination in the E.coli, which include two recombinations—retrieve and neo knock-in, we got three gene targeting vectors of Resp18 gene with different length of homologous arms. Then, we chose the targeting vector-- pBR322-Resp18R-20KO ,which have the longest homology arm, to target the ES cell. The clones survived in the culture ES cell media with G418 and Ganciclovir were confirmed to have recombination at the expected sites by PCR and realtime PCR. Later, we have the positive clones microinjected into the blastocyst prepared and transplanted the blastocyst into pseudopregnancy female mice. The offsprings of these pseudopregnancy mice were chimeric mice. And got the heterozygote mice by chimeric mice intercrossing with C57BJ/6L mice. Further descend breeding by mating of heterozygote mice, we can get three types of Resp18KO mice including homozygote,heterozygote and wild type.We start to observe the phenotype of these mice since we got the heterozygote mice, including condition of gestation,breeding,spirit,action,foodintaking. Finally, according to the expression pattern of Resp18 in different tissures, we further analyses the phenotype of Resp18KO mouse model.Result: We obtained gene targetting vector of Colec11 by the method of ET clone technology successfully, and establish the platform of ET clone technology.Furthermore, in the study of gene Resp18,the efficiency of retrieve and neo knock-in in the ET recombination is about fifty pencent and above ninety percent. After microinjection of Resp18 gene targeting vector with the longest homology arms into ES cells, the efficiency of homologous recombination is about ten percent. And we got eight male mice with more than fifty percent of gomphosis after transplant of the blastocyst. We obtain twenty-three heterozygote mice from the offsprings of the chimeric mice, and no homozygote mice were identified in the offsprings of heterozygote mice yet.Conclusion: The success of construction of gene targetting vector not only provide the base for the futher investigation of the function of Colec11 gene, but also proved the high efficiency and practicality of ET clone technology in the construction of gene targeting vector. ET recombination is a high efficent method in the constuction of gene targeting vector, which can easily retrieve homology arm within 20 kb from BAC DNA without any mutation. This method also insure the high efficency of homologous recombination. As a secreted protein, Resp18 has certain function in the growth and development of mice,especially in the process of embryo development,which may lead to the lethal of homozygote mice. The analyses the phenotype of Resp18KO mouse model is still in the progress.
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