Two Gene Constructs Contained Different Fragments Of SV40T And Establishment Of Transgenic Mouse

Simian Virus40(SV40) is the small DNA tumor virus, which belongs to Papoviruidae. SV40 has the characteristics to disrupt the control of host cell growth by interacting with cellular proteins and to induce tumorigenesis. The characteristic of transforming activity is mostly related to T antigen-early protein. So many researches showed that most SV40T transgenic mice could be induced many kinds of tumors, which suggested that SV40T transgenic mice can be used for the researches of the exploration of tumorigenesis mechanisms.To study the relationship between function ofRb,p53 genes and the tumorgenesis of SV40T, two genes contained different fragments of SV40T were constructed: one gene is called SV40T-DRb which was mutated in Rb motif of SV40T, the other gene is called SV40T-Dp53 which was deleted p53-binding domain. Then it will be useful for discussing the function of p53 and Rb gene in the tumorgenesis related to SV40T to establishing SV40T-DRb and SV40T-Dp53 transgenic mice. Findings of this research are following: 1. Using gene cloning techniques of molecular biology, the changed two gene fragments of SVOT were cloned and sequenced. The results showed that there isonly one mutation in 109th amino acid(E-V) of Rb-binding domain in SV40T-DRb, there are three mutations happened in SV40T-Dp53: two mutations cause the change of amino acid, but the two mutations are outside from Rb-binding domain; one mutation does not cause the change of amino acid. 2. Finally the two genes were cloned into the galactophore-specific expressed vector-p205C3 respectively. Using microinjection into male nuclei, two kinds of transgenic mice were established. In this research, total 862 mice embryos were microinjected, 360 mice embryos were survive, 35 mice receptors were used, 23 female mice were pregnant, 127 mice were born. 3. Using PCR, six transgenic mice were detected positive for both gene SV40T-DRb and gene SV40T-Dp53. To confirm the positive transgenic mice, southern blot was used to detect positive transgenic mice. The result showed one transgenic mouse( ) that was positive for both gene SV40T-DRb and gene SV40T-Dp53. 4. Total 25 F1 mice were born from FO positive transgenic mouse, the positive rate is 50%( 16/25) after PCR detection, which suggested that this was conformed to the laws of Mondel inheritance and transgene could be passaged to F1.These suggested that the two gene constructs contained different fragments are successful and the transgenic mice model will become strongly useful tool to study the relationship ofRb,p53 genes and SV40T gene.