Experimental Studies On Generation Of Transgenic Mice By Intratesticular Injection And In Vivo Electroporation

Transgenic animal have extensive perspective of research and application in field of medicine, life science and biopharmacy, for this reason, scientists had been explored a more convenient and easy, economic and practice, and high-performance transgenic methods. Recently a few years, with the development of the science and technology, in vivo gene transfer have become a popular tool to be used for the fields of the gene therapy, biological analysis. In vivo electroporation (EP) is one of these.Spermatogenic cell is spermatic precursor cell in male animal and the only cell type in postnatal mammals which has the capability to self-renew and to contribute genetic information to the next generation. The manipulation of spermatogenic cells and modification of their genomes have great significance in building transgenic animal model, preparation mammary gland bioreactor. in order to explore the feasibility of constructing transgenic animal by exogenous gene are injected into seminiferous tubule of testis of living animal, we have designed two groups different in vivo EP parameter of low voltage (30~110V/cm) and high voltage(500~900V/cm) , they were used for in vivo EP of testis of SPF male KM mice, which observe effect of damage degree and reproductive capability for testis of SPF male KM mice in different EP parameter, its purpose is screened suitable in vivo EP parameter. Based on this condition, we carried out two different treatments for eukaryon expression plasmid pCE-29 DNA which inserted enhanced green fluorescent protein (EGFP) : (1) pCE-29 DNA +transfection reagent (in vivo jetPEI?) +trypanblue(TB); (2) pCE-29DNA+ TB. Then these different treatment pCE-29 DNA were injected into seminiferous tubule of testis of SPF male KM mice to be divided into four groups respectively, among two groups of injection with pCE-29 DNA +transfection reagent (in vivo jetPEI?) + TB, one group was performed in vivo EP, another group did not it; another two groups of injection with pCE-29 DNA + TB were performed the same treatment yet, which observe whether it has difference between in vivo EP and non-EP or not for different treatment exogenous interest gene that were injected into seminiferous tubule. Thereby, it can provide a convenient and high efficient method for in vivo gene transfer. SPF male KM mice after two weeks mated with SPF female KM mice by superovulation respectively, until the new litters were born. Genomic DNA of new born litter tail were extracted and run PCR-Southern detection, in order to confirm integration and expression efficiency of exogenous gene that were treated in four different treatment means. These results show: (1) two weeks after in vivo high voltage EP through500~900V/cm, it made irreversible damage of germ cell in seminiferous tubule of SPF male KM mice, and resulted in loss of reproductive capacity of SPF male KM mice; In addition, weight of testis, spermatozoa viability of epididymis and shape & construction of testis have significantly degrade and chang to compare with normal testis of mice(p<0.05). (2) two weeks after in vivo low voltage EP through 30~ HOV/cm, although it will make some damage of testis of SPF male mice, this damage is reversible, seminiferous tubule of testis were able to resume for some time and still produce new spermatid, SPF male mice have reproductive capacity. Meanwhile, we screened a suitable in vivo EP parameter combination by evaluation of spermatozoa viability of epididymis, damage degree of seminiferous tubule and mating test with female. (3) we chosen 20 from Fl new born offspring in four groups of different treatment respectively, then, genomic DNA were extracted from tail of Fl offspring, extractive genomic DNA were used for PCR-Southern detection, the results indicate: it has significantly difference that integration efficiency of exogenous interest gene of Fl new born offspring between different groups, integration ratio of foreign gene of group A (25 %) is the most high among the four groups, group C is 10 %, group B is 5%, group D is 0%(p<0.05). Meanwhile, it is high than microinjection (integration ratio: pig 10%, sheep 1.4% -21.2%, cattle 5.6% ) and retrovirus (mice: 4.5%).It is concluded from this study that we have constructed a new method whichgenerate transgenic mice through recombination means of intratesticular injection and in vivo EP. This method compares to retrovirus (preparation complication of retro virus) and microinjection(requirement special instrumentation, proficient operator, procedure of complex operation), it has many advantages which are convenient operation, requirement less of instrumentation, high transfection ratio and easy delivering to the next generation, it opened a new pathway for producing of transgenic mice and in vivo gene transfer.