Studies On The Production Of Phytase Transgenic Pigs By Handmade Cloning

Rapidly developing and intensive animal husbandry of livestock is a major contributor to global environmental pollutions.Phytase can not be secreted by the monogastric animals such as pig and chicken.Large quantities and high concentrations of manure waste which contains phytate phosphorus are generated,which leading to environmental pollutions.So it is important to economically utilize the environmental protection and phosphorus resources by improving the utilization rate of phytic acid phosphorus in feed.Phytase is a kind of hydrolase.Phytic acid can be decomposed into inositol and phosphoric acid by phytase,which could be digested and absorbed by animals.It can effectively increase the phytic acid phosphorus utilization in the diet,and reduce fecal phosphorus emissions,so as to reduce the phosphorus pollution to the environment.With the development of genetic engineering technology,especially the improvement on the level of production technology on transgenic animals,there is an effective new way to solve these problems.Endogenous phytase with high biological activity was produced through endogenous digestive tract of the monogastric animals such as pigs and poultry,which has been a research strategy and method to solve the high phosphorus pollution in the fecal material of the monogastric animals,and a kind of method for once and for all as well.The study combined a phytase gene which was insensitive to cleavage by pepsin and trypsin and also had a higher affinity to the substrate with the pig parotid secretory protein(PSP)gene promoter.The tissue specific vectors p-PSP-Intron-Cafp and PSP-Cafp-IRES-EGFP were constructed and PSP-Cafp-IRES-EGFP was expressed in Parotid gland cells in the mice.Embryo culture platform was optimized through the project.The matured medium without FBS was better than the medium with FBS after culturing 22-24h.Pzm-3 medium was better than NCSU medium for culturing embryo.It was also good when culture medium with NCSU adding 10%FBS at 4th day.The parameter with 120V,30?s,3 pulse was the best for the parthenogenesis.Two transgenic positive fetal fibroblasts cell lines DB and LW indentified by PCR with p-PSP-Intron-Cafp vector were abtained by electroporation.The cell lines carrying Cafp were used as nuclear donors in handmade cloning.Cloned embryos were cultured in vitro to blastocysts by handmade cloning.We generated 810 blastocysts and 712 were of good quality and transferred to 6 recipients,of which 4 were successful pregnant and 3 were delivered.Fourteen piglets were born of which 3 delivered.The PCR and sequencing results showed that 7(3 live and 4 dead)of the 14 piglets carried Cafp.There were 5 positive piglets among 6 survived piglets in Fluorescence in situ hybridization.All the 6 survived piglets were positive in polyacrylamide gel electrophoresis while none of them were positive when tested by mass spectrometry.Phosphorus digestion rate of the 6 live cloned pigs and 3 pigs for control was tested at 6 months of age.The Phosphorus digestion average rate in transgenic positive group was 57.17%,10 percent higher than the average rate in control group(44.7%)with significant difference(P<0.05)and the average rate in transgenic negative group was 47.2%.IHC results on the 3 dead pigs after 6 months,the Cafp positive pig showed that the transgene expressed only in parotids,confirming tissue-specific gene expression and the function of the promoter PSP.In conclusion,Cleavage-resistant phytase transgenic pigs were successfully produced through handmade cloning.The cloned pigs offer a unique biological approach to manage phosphorus nutrition and environmental pollution in animal husbandry.