Lay Jean M, Bane Gina, Brunkan Cynthia S, Davis Jennifer, Lopez-Diaz Lymari, Samuelson Linda C
Department of Molecular and Integrative Physiology, The University of Michigan, 7761 Med Sci II, Ann Arbor, MI 48109-0622, USA.
Am J Physiol Gastrointest Liver Physiol. 2005 Feb;288(2):G354-61. doi: 10.1152/ajpgi.00356.2004. Epub 2004 Oct 14.
CCK is predominantly expressed in subsets of endocrine cells in the intestine and neurons in the brain. We evaluated the expression of a CCK gene construct in transgenic mice and cultured cells to identify a genomic region that directs correct tissue- and cell-specific expression in enteroendocrine cells. The CCKL1 transgene contained 6.4 kb of mouse Cck fused to lacZ. Expression was evaluated in three transgenic lines (J11, J12, J14) by measurement of beta-galactosidase in tissue homogenates and frozen sections. Correct tissue-specific expression was observed, with beta-galactosidase activity detected in intestine and brain. However, there were differences seen in cell-specific expression in the intestine. Line J14 exhibited expression in CCK-endocrine cells, with expressing cells arising at the normal time during fetal development. However, transgene expression in line J12 intestine was limited to neurons of the enteric nervous system, which reflect an early fetal expression pattern for CCK. Analysis of an additional 15 transgenic founder mice demonstrated intestinal expression in 40% of transgenics, with expressing mice following either an endocrine cell pattern or a neuronal pattern in approximately equal numbers. CCKL1 transfection analysis in cultured cells also demonstrated enteroendocrine cell expression, with 100-fold enhanced activity in the enteroendocrine cell line STC-1 compared with nonendocrine cell lines. The results suggest that the minimal cis-regulatory DNA elements necessary for appropriate CCK expression in enteroendocrine cells reside within the 6.4-kb mouse genomic fragment.
胆囊收缩素（CCK）主要在肠道内分泌细胞亚群和大脑神经元中表达。我们评估了CCK基因构建体在转基因小鼠和培养细胞中的表达，以确定一个指导肠内分泌细胞中正确组织和细胞特异性表达的基因组区域。CCKL1转基因包含与lacZ融合的6.4 kb小鼠Cck。通过测量组织匀浆和冰冻切片中的β-半乳糖苷酶，在三个转基因品系（J11、J12、J14）中评估表达情况。观察到了正确的组织特异性表达，在肠道和大脑中检测到了β-半乳糖苷酶活性。然而，在肠道的细胞特异性表达上存在差异。J14品系在CCK内分泌细胞中表现出表达，表达细胞在胎儿发育的正常时间出现。然而，J12品系肠道中的转基因表达仅限于肠神经系统的神经元，这反映了CCK的早期胎儿表达模式。对另外15只转基因奠基小鼠的分析表明，40%的转基因小鼠在肠道中有表达，表达小鼠中内分泌细胞模式和神经元模式的数量大致相等。在培养细胞中的CCKL1转染分析也证明了肠内分泌细胞的表达，与非内分泌细胞系相比，肠内分泌细胞系STC-1中的活性增强了100倍。结果表明，肠内分泌细胞中CCK适当表达所需的最小顺式调控DNA元件位于6.4 kb的小鼠基因组片段内。
