Studies On The Transgenic Silkworm Mediated By Non-transposon Vector

The silkworm (Bombyx mori) has been a model organism for classical geneticsresearch because of its short reproduction cycle, highly reproductive rate, well-knowninheriting background, large amount of offspring and can be raised on a large-scaleartificially. Transgenic silkworm may generate new variety and heterologous protein,from which people enjoy a huge potential for development and a great value inapplications. The kinds of vector of gene transfer for producing transgenic silkwormmainly cover transposon vector and non-transposon vector, of which, non-transposonvector has been applied rarely. The purpose of our research is to try transgenic silkwormmediated by non-transposon vector so as to widen methoeds for application oftransgenic silkworm.Human insulin-like growth factor-I (hIGF-I) is a multifunctional proliferationcontrolling factor of cells. hIGF-I can stimulate the growth and differentiation of cells invitro and in vivo. It is the main regulator of growth hormone, and has the function ofreducing blood sugar similar to insulin. Therefore it has a broad application perspectivein the field of medical research. Since the low content and great purification difficultiesof natural hIGF-I in human blood, the insufficient supply has become the main obstaclefor its research and application. Further more, as a high eukaryotic expression system,Silkworm shows a great application prospect among production of genetic engineeringdrugs. To confirm the possibility of foreign gene expression in transgenic silkwormmediated by non-transposon vector pIZT/V5-His, the hIGF-I gene was cloned intovector pIZT/V5-His to generate transgenic vector pIZT/V5-His-hIGF–I, the vector wasthen introduced into the eggs of silkworm by sperm-mediated method. Transgenicsilkworms were obtained by verification with fluorescence observation, PCR and Dotblotting, and were reared to the G2generation. Western blotting analysis using anantibody against hIGF-I demonstrated a specific band with a molecular weight of 12.5kD in the transgenic silkworms. meanwhile, the expression level of hIGF-Idetermined by ELISA assay was approximately65,411,469ng/g of freeze-driedpowders of whole bodies, posterior silk glands and fat bodies, respectively. indicatingthat a heterologous gene could be integrated into silkworm genome by non-transposonvector and expressed successfully.On the other hand, to explore the transgenic silkworms based on gene targetingtechnique, we use the fragments upstream1.2kb and downstream1.2kb of the ovo geneas homogenous arms, then the gfp gene driven by a3promoter, the neo gene driven byie-1promoter were inserted into the gap between the two homologous arms as thepositive chosing factor, the ie-1-driven DsRed gene was cloned at downstream of theright homologous arm as the negatively selecting factor. Then the targeted vectorpSK-ovoL-A3-GFP-IE-neo-ovoR-IE-DsRed was obstained. The vector was thenintroduced into the eggs of silkworm by sperm-mediated method. Transgenic silkwormswere obtained by the verification with G418, fluorescence observation, and PCR.Further study on the integration site of the heterologous genes shows that the integrationis random, the obtained transgenic silkworms are non-homologous recombinationtransgenic silkworms based on gene targeting vector, also demonstrates the lowefficiency of homologous recombination.