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.

The advent of genetically engineered pig production has revealed a wide array of opportunities to enhance both biomedical and agricultural industries. One powerful method to develop these models is transgenesis; however, selection of a suitable promoter to drive transgene expression is critical. The cytomegalovirus (CMV) promoter is the most commonly used viral promoter as it robustly drives transgene expression in a ubiquitous nature. However, recent reports suggest that the level of CMV promoter activity is tissue-dependent in the pig. Therefore, the objective of this study was to quantify the activity of the CMV promoter in a wide range of porcine tissues. Swine harboring a CMV-ZsGreen1 transgene were utilized for this study. Thirty five tissue samples were collected from neonatal hemizygous (n = 3) and homozygous (n = 3) transgenic piglets and analyzed for ZsGreen1 abundance via immunoblot. ZsGreen1 was detected in all tissues examined; however, quantification revealed that the level of ZsGreen1 expression was tissue-specific. For example, ZsGreen1 was most abundantly produced in the salivary gland, moderately produced in the esophagus and lowly expressed in the stomach. Interestingly, expression of ZsGreen1 also differed within organ. For instance, levels were highest in the right ventricle compared with other chambers of the heart. The expression patterns of ZsGreen1 were similar between homozygous and hemizygous piglets. Ultimately, these results elucidate the tissuespecific activity of the CMV promoter in the neonatal pig.All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.