Preliminary Studies On Sperm-mediated Gene Transfer Technique In Domestic Silkworms

In order to understand the efficiency of sperm-mediated gene transfer in domestic silkworms and the influence of foreign DNA's concentration, as well as its conformation on the gene transfer in domestic silkoworms, the homogenously recombinant expressing GFP plasmid pG350, and an EGFP expression vector, which was derived from pG350 and pMD18-T, were employed along with pEGFP-N3 as the transgenic vectors.During the past 3 years, 2 independent experiments of sperm-mediated gene transfer in domestic silkworms were carried out respectively in 2000 and 2002 with the method direct injection of copulation thecae.In 2000, linear pG350 with the concentration of 2.0 mg/mL was used in the gene transfer experiment in silkworms. 5 of 14 DNA samples extracted from GO spawns were positive by PCR detection (positive proportion 35.7%), and the results of PCR and blotting detection of GI silkworms indicated that the gfp gene is in the genome of Gj silkworms.In 2002, two plasmids (pMD-Fib-IE-EGFP and pEGFP-N3) were used in the gene transfer experiment of domestic silkworms. pMD-Fib-IE-CGFP was transferred in 3 different concentrations (2.0 1.4 0.7mg/mL) and 2 conformations( linear and circular) respectively corresponding to group X20, X14, X07, H20, H14, H07; Circular PEGFP-N3 was transferred with the concentration 1.2 mg/mL(corresponding to group H12).The PCR positive proportions of group X20, X14, X07, H20, H14, H07,H12 were respectively 55.0%, 78.6%, 53.3%, 50.0%, 58.3%, 71.4%, 46.2%. The hybridization results indicated that the egfp gene has been integrated in the genome of Go silkworms.There was not any distinct difference in flourrescence density between the experimental and control silkworms under the ultra-violet light in both of the 2 experiments; and with the excitation of ultra-violet ray, the germline cells of experimental silkworms in the experiment of 2002 emitted green fluorescence while the control did not.The research demonstrated that the sperm-mediated gene transfer method (direct injection of copulation thecae) can introduce foreign gene into silkworms, and that the foreign gene may be integrated in the chromosomes of silkworms with many copies in series and can be transmitted to G1 offspring.