Desaulniers Amy T, Cederberg Rebecca A, Carreiro Elizabeth P, Gurumurthy Channabasavaiah B, White Brett R
Department of Animal Science, University of Nebraska-Lincoln, Lincoln, NE 68583-0908, USA.
Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5930, USA.
J Biomed Res. 2020 Dec 25;35(2):163-173. doi: 10.7555/JBR.34.20200111.
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 ( =35) were collected from neonatal hemizygous ( =3) and homozygous ( =3) piglets and ZsGreen1 abundance was determined immunoblotting. ZsGreen1 was detected in all tissues, except hypothalamus, kidney cortex and oviduct. The expression patterns of homozygous and hemizygous piglets were similar ( >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 ( <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 ( <0.05). Interestingly, ZsGreen1 amounts also differed within an organ ( , the right ventricle had 3-fold higher levels than the other heart chambers; <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.
由于猪的基因工程对生物医学和农业都有益处，因此选择合适的基因启动子至关重要。巨细胞病毒（CMV）启动子能够强有力地驱动转基因的普遍表达，目前被广泛使用，但最近的报告表明其在猪体内具有组织特异性活性。本研究的目的是对具有单个整合位点的CMV-ZsGreen1转基因猪的组织中的ZsGreen1蛋白（代替CMV启动子活性）进行定量分析。从新生的半合子（n = 3）和纯合子（n = 3）仔猪中采集组织样本（n = 35），并通过免疫印迹法测定ZsGreen1的丰度。除下丘脑、肾皮质和输卵管外，在所有组织中均检测到了ZsGreen1。纯合子和半合子仔猪的表达模式相似（P>0.05）。然而，定量分析表明ZsGreen1蛋白水平具有组织特异性。在神经/内分泌组织中，ZsGreen1丰度在前垂体中最高，在小脑中居中，在大脑、脊髓和后垂体中最低（P<0.05）。在消化系统中，ZsGreen1在唾液腺中的含量高于食管、胃、胰腺、十二指肠、空肠、回肠、脾脏、结肠、胆囊和肝脏（P<0.05）。有趣的是，ZsGreen1的含量在一个器官内也有所不同（例如，右心室的水平比其他心腔高3倍；P<0.05）。这些结果为使用CMV启动子驱动猪体内的转基因表达提供了有用信息。此外，这种猪模型代表了一种新的ZsGreen1标记器官资源，也是推进基因组编辑研究的有价值工具。
