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Production of cleavage-resistant phytase transgenic pigs by handmade cloning

Zhang Ming-yuan  Yang Hao-wen  Guo Xiao-ping  Huang Yun  Qi Li-na  Chen Xiao-liang  Wei Ling-jing  Chen Bao-jian  Chen Qiu-ming  Wu Yan-jun  Jiang Qin-yang  Guo Ya-fen  WeiYing-ming  Lin Xiu-kun  Jiang He-sheng  Lan Gan-qiu  
【摘要】:Rapidly developing and intensive animal husbandry of livestock is a major contributor to global environmental pollutions.Large quantities and high concentrations of manure waste which contains phytate phosphorus are generated.The use of phytase can effectively solve the problem of high phosphorus pollution in the fecal material of monogastric animals.Enviropigs,producing phytase in the salivary glands and secreting the enzyme in the saliva were first generated at the University of Guelph in 1999.However,this enzyme is easily inactivated in digestive processing.To address this problem,we improved the transgene construct and successfully generated cleavage-resistant phytase transgenic pigs by handmade cloning(HMC).Three cell lines carrying Cafp were obtained.Through handmade cloning,810 blastocysts on Day 6 were generated and 712 were of good quality and were transferred to six recipients.Fourteen piglets were born of which six survived.The PCR and sequencing results showed that seven(three live and four dead) of the 14 piglets carried Cafp.Phytase activity in the saliva of the six live cloned pigs was tested at four months of age and only one pig had 0.155 FTU/ml enzyme activity.The other two may not be activated in the transgenic parotid gland.Among all the transgenic pigs,the highest phosphorus digestion rate was 59.2% of intake,which represents a 25.4% decrease in fecal emission compared to the average of controls.IHC results on the three later dead Cafp positive pigs showed that the transgene expressed only in parotid glands,confirming tissue-specific gene expression.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.

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