Generation Of Swine PID1Overexpression Transgenic Rab-bits By Testicular Injection

With the growth in the living standard, people’s demanding for the quality of pork isgetting higher and higher. Intramuscular fat (IMF) content is an important influencing factorfor meat, it is correlated with the tenderness, juiciness and flavor of meat. From last century,many producers and researchers are focusing on the production of an edible lean meat with aminimum of visible fat. So meat quality has been showing a steady decrease, which can beseen in decreasing intramuscular fat content (IMF) values. And now the IMF has dropped toabout1.5%in the commercial breeds, which has a serious impact on the taste of meat. Theprevious studies of our lab found that the PID1gene affects intramuscular fat contentsignificantly. The PID1gene expresses highly in Laiwu pig, which possesses a high IMFcontent. Many researchers have confirmed the effect of this gene on IMF in pig. In this study,we injected the expression vector pIRES2-acGFP-PID1carrying the pig PID1gene into thetestis of the New Zealand male rabbits to generate a transgenic animal model. We examinedthe F1and F2generations to further investigate the relationship between the PID1gene andvariation in IMF content and the possibility to generate transgenic animals by testicularinjection. The main results are as follows:(1) We cloned the full-length of the CDS of the PID1by PCR using a pair of specificprimers, whose5’end has been added a restriction endonuclease Bgl II and EcoR Irespectively and the protective bases joined too. To build the pIRES2-acGFP-PID1plasmid,the expression vector pIRES2-AcGFP1and the PID1gene were digested, recovered, purifiedand then ligated with the T4ligase. At last, we tested the recombinant plasmid throughdigestion and sequencing.(2) Six healthy adult New Zealand male rabbits were selected, and they were treated bybilateral testicular injection. The injection composed of the pIRES2-acGFP-PID1, transfectionreagents (X-treme GENE HP from Roche) and appropriate DMEM (The former two havebeen incubated15minutes before mingled with the latter). Another two rabbits were selectedas the control group, and they were also treated by bilateral testicular injection except theinjection contained same amount of saline only. The injection continued three weeks with onetime for each week. One month after the last injection, all of the eight male rabbits mated with normal females at a ratio of1:6to get the F1generation. Then the F1hybridized to get the F2generation.(3) In our experiment, the test group gave birth to170F1offspring (61transgenicpositive ones and109negative ones) with a transgenic positive rate of35.88%. The controlgroup gave birth to48offspring. The F1generation gave birth to67F2offspring (23transgenic positive ones and44negative ones) with a transgenic positive rate of34.33%. Thecontrol group gave birth to29offspring.(4) We measured all the meat quality parameters of the F1generation. Our analysisdemonstrated that the IMF content was elevated significantly in the F1transgenic positiveindividuals compared to the negative ones and the control group (P<0.05). The daily gain andthe pH24hof the F1transgenic positive ones were significantly higher compared to the controlgroup, while no differences were found between the transgenic positive individuals and thenegative ones. The eviscerated weight, drip loss, tenderness and SOD24hof the test groupdiffered significantly with the control group (P<0.05). The dry matter, half-evisceratedweight, pH45min, meat color, T-AOC and MDA were no differences in the three groups. Theprotein expression of the PID1was higher in the F1transgenic positive individuals than theother two groups. The fluorescence of the transgenic positive rabbits was more intense thanthe control group, and it fluoresced in the limbs and tail mainly.(5) We measured the meat quality of the F2generation, too. The IMF content of the F2generation was elevated significantly in the transgenic positive individuals compared to thenegative ones and the control group (P<0.05). The daily gain of the F2transgenic positiveones and negative ones were significantly higher compared to the control group, while nodifferences were found between the transgenic positive individuals and the negative ones.Drip loss of the positive individuals differed significantly with the control group (P<0.05),while no differences were found in other groups. The dry matter was no differences in thethree groups.In this study, we produced transgenic rabbit carrying pig PID1gene by testicularinjection successfully. Meat quality traits, which included IMF, tenderness, drip loss, declinerate of the pH and protein expression were improved in the transgenic rabbits. But the growthrate was slower and the antioxidant capacity of muscle declined.