Since genetic engineering of pigs can benefit both biomedicine and agriculture,selecting a suitable gene promoter is critically important.The cytomegalovirus(CMV)promoter,which can robustly drive ubiquitous transgene ...Since genetic engineering of pigs can benefit both biomedicine and agriculture,selecting a suitable gene promoter is critically important.The cytomegalovirus(CMV)promoter,which can robustly drive ubiquitous transgene expression,is commonly used at present,yet recent reports suggest tissue-specific activity in the pig.The objective of this study was to quantify ZsGreen1 protein(in lieu of CMV promoter activity)in tissues from pigs harboring a CMV-ZsGreen1 transgene with a single integration site.Tissue samples(n=35)were collected from neonatal hemizygous(n=3)and homozygous(n=3)piglets and ZsGreen1 abundance was determined via immunoblotting.ZsGreen1 was detected in all tissues,except hypothalamus,kidney cortex and oviduct.The expression patterns of homozygous and hemizygous piglets were similar(P>0.05).However,quantification revealed that ZsGreen1 protein levels were tissue-specific.Within neural/endocrine tissues,ZsGreen1 abundance was highest in the anterior pituitary gland,intermediate in the cerebellum and lowest in the cerebrum,spinal cord and posterior pituitary(P<0.05).In the digestive system,ZsGreen1 was more abundant in the salivary gland than esophagus,stomach,pancreas,duodenum,jejunum,ileum,spleen,colon,gallbladder and liver(P<0.05).Interestingly,ZsGreen1 amounts also differed within an organ(i.e.,the right ventricle had 3-fold higher levels than the other heart chambers;P<0.05).These results provide useful information for the use of the CMV promoter to drive transgene expression in the pig.Moreover,this swine model represents a novel resource of ZsGreen1-labeled organs and a valuable tool to advance genome editing research.展开更多
基金This project was partially supported by the Nebraska Agricultural Experiment Station with funding from the Hatch Multistate Research capacity funding program(Accession No.1011129 to BRW)an Agriculture and Food Research Initiative(AFRI)Competitive Grant(2017-67015-26508 to BRW)aan AFRI ELI predoctoral fellowship(2017-67011-26036 to ATD)from the USDA National Institute of Food and Agriculture.The funding agency had no role in study design,data collection,interpretation,or manuscript generation.The authors wish to thank Ginger Mills for pig husbandry as well as Scott Kurz,Guilherme Cezar,Kyle Regan and Amanda Lambrecht for technical assistance.The authors would also like to express gratitude to Lelanya Yates for aiding in the dissections.
文摘Since genetic engineering of pigs can benefit both biomedicine and agriculture,selecting a suitable gene promoter is critically important.The cytomegalovirus(CMV)promoter,which can robustly drive ubiquitous transgene expression,is commonly used at present,yet recent reports suggest tissue-specific activity in the pig.The objective of this study was to quantify ZsGreen1 protein(in lieu of CMV promoter activity)in tissues from pigs harboring a CMV-ZsGreen1 transgene with a single integration site.Tissue samples(n=35)were collected from neonatal hemizygous(n=3)and homozygous(n=3)piglets and ZsGreen1 abundance was determined via immunoblotting.ZsGreen1 was detected in all tissues,except hypothalamus,kidney cortex and oviduct.The expression patterns of homozygous and hemizygous piglets were similar(P>0.05).However,quantification revealed that ZsGreen1 protein levels were tissue-specific.Within neural/endocrine tissues,ZsGreen1 abundance was highest in the anterior pituitary gland,intermediate in the cerebellum and lowest in the cerebrum,spinal cord and posterior pituitary(P<0.05).In the digestive system,ZsGreen1 was more abundant in the salivary gland than esophagus,stomach,pancreas,duodenum,jejunum,ileum,spleen,colon,gallbladder and liver(P<0.05).Interestingly,ZsGreen1 amounts also differed within an organ(i.e.,the right ventricle had 3-fold higher levels than the other heart chambers;P<0.05).These results provide useful information for the use of the CMV promoter to drive transgene expression in the pig.Moreover,this swine model represents a novel resource of ZsGreen1-labeled organs and a valuable tool to advance genome editing research.
