The Integration Of Three Human DNA Fragments Coinjected Into Mice

Transgenic animal is a new-born bio-technique, which is the development and extension of general molecule biology and gene engineering. With the appearing of transgenic animal, it is possible that people can rebuild the genome of animals by gene recombination and contrive research on relative gene's structure and function in viviperception. This is a multidimension system of gene research with regard to providing new approaches and thoughts from molecule to individual arrangement. Transgenic animal reflects the complexity of nature, opening up avenues of life research previously impenetrable, and having a splendid application as well.The approach of gene coinjection was firstly used by Clark's research team in Britain, aiming to improve the expression level of foreign gene by its cooperating action. Thereafter, it has been increasingly using to find out the impact of gene and its elements on expression. By now, it has been successfully used to produce transgenic mice which contain two or three genes in vivo.In this paper, three human DNA fragments were coinjected into mice in order to study the influence of uncoding large DNA fragment on HSA or hFEN1 gene's integration efficiency, to explore methods to produce high expression of human serum album animals, to find out how to establish multi-transgenic animals in a way of saving time and money, to provide experience and reference to others. It mainly contains the following aspects:Firstly, according to the sequence of human serum album gene cDNA in GeneBank, one primer was designed. Secondly, with human's liver tissue DNA as templates , the gene fragment of HSA was amplified by PCR. Then PCR products were subcloned into pGEM-T vector and transformed E.coli.host strains. Correct clones were selected and plasmid DNA was isolated and digested with SacI and PuvII .A DNA fragment of About 2.1kb was purified and labeled by DIG-11dUTP as probe. At least 40 positive clones were obtained from human genomic library by in situ colony hybridyzation with this probe. Among them one clone contains human serum album DNA by sequence. And we select two other positive clones, one contains DNA repair gene-hFEN1 and the other CIT987SK-384D8-an uncoding large DNA fragment. They were coinjected into the male prenuclei of fertilized eggs with HSA. DNA together . After that normal injected eggs were selected and transferred to the oviduct of pseudopregment recipents mice and gave birth to 65 Fl offsprings, the foreign genes were found integrated in 12 of 65 mice by PCR and Southern blotting detection. The total efficiency of coinjection was 2.08%. This study obtained the results and advances as follows: 1. twelve transgenic mice were successfully produced by coinjection with three human DNA fragments-HSA DNA , hFENl and an uncoding CIT987SK-384D8. 2. The efficiencies of HSA and hFENl integration have no mutually influences, and both can be improved by the uncoding large DNA fragment. 3. It is applicable to coinject two or three genes whose expression products have no antagonistic action. 4. Coinjection is a simple gene transfer method which saves time and money.