| In the male sex determination is switched by a single gene on the short arm of the Y chromosome, named as SRY. SRY gene belongs to a family of High Mobility Group (HMG),encodes a protein with an HMG-like DNA-binding domain, which probably acts as a local organizer of chromatin structure. It is the discovery of Sry that let study of sex controlling be on the right way. However, so far most transgene investigation, especially on Sry, only limit on few laboratories due to its restricted requirements. On the other hand, the transgene method mediated by sperm appears extremely easy to use in all sexual reproductive animals with obvious advantage of low costing, high efficiency (>10%), less equipments needed, no mechanical invasion for oocyte, similar to natural fertilization. However, almost all the recent studies were focus on the group of genes with closed related to a few enzymes in the crucial metabolism and the advantages of sperm mediated transgene was not highlighted. So our experiments were firstly designed, to our knowledge, for Sry transgene mediated by sperm to investigate whether it could shift sex of the offspring. Not only is it important for sex developing induced by Sry, but also open a new potential way of sex controling in animal production.The target Sry was amplified by PCR from mouse DNA samples extracted from blood cells with specified primers designed according to the sequences on database of Genbank. After cloning into vector of pMD18-T ,tbe acquired Sry fragment was sequenced for conforming on Genbank. Then Sry were cut off by restricted enzymes with two sectioned points (EcoR I /Not I), and then recombined into an eucaryote expression vector pCR3.1.Sperms were subjected to examination of fluorescent in situ hybridization (FISH) with specific probes for verifying the position the Sry intergranted. Before the incubation of Sry plasmid with the sperms the sperms samples were divided as washing and unwashing groups in order to check if the washing might enhance the efficiency of Sry gene absorption. The microscopic observations shown that about 50% sperms that weren't done any disposal have positive signal located into or around the region of the karyoplasms. the sperm-mediated Sry ,the un-washed the positivesignal outside the karyoplasms,lie in its circumjacent districts of tail or the head, on the contrast, the washed most can catch the vision of the positive signal located into the karyoplasms then hided under the karyoplasms or recombined into the chromatin, especially added liposome. Therefore, the phenomena support the idea that there may be some factors in the semen that inhibits absorption of DNA into sperms. This observation is very important for helping our knowledge about the sperm mediated transgene investigation. The pictures from fluorescence in situ hybridization all shown that the result could promote the further awareness of Sry and the accurate site of the transgened one,in addition, provide more information to the understanding of the transgened one.In the animal experiments we applied in vitro fertilization for the mouse reproduction due to the requirement of in vitro manipulation of the semen. This may have general influence upto the rate of fertilization. As the compensatory we promoted the reproductive efficiency by treating the animal with PMSG and HCG. Compared with natural copulation the litter size decreased in all the different treatment, including sham treatment. They were 44.5,45.3,48.3,38.1,40.8 percent of control group respectively. This expected results only means a general reproductive efficiency lowered by in vitro procedure of semen but nothing on the sex differentia. With careful controlling the transgene efficiency of different methods was monitored by sex ratio of offspring. Our data shown that the highest rate of Sry gene transfer is groups of washed sperms plusing liposome (1.842) , then only washed sperms(1.203) respectively. As reported our data also shown that liposome could elevate ability of sperm absorption for target DNA. Unfortunate...
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