Transgenic mice containing intestinal fatty acid-binding protein-human growth hormone fusion genes exhibit correct regional and cell-specific expression of the reporter gene in their small intestine.
Transgenic mice containing intestinal fatty acid-binding protein-human growth hormone fusion genes exhibit correct regional and cell-specific expression of the reporter gene in their small intestine.
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含有肠脂肪酸结合蛋白-人生长激素融合基因的转基因小鼠在其小肠中表现出报告基因的正确区域和细胞特异性表达。
DOI：
10.1073/pnas.85.24.9611
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发表时间：
1988
影响因子：
11.1
通讯作者：
Gordon,JI
中科院分区：
文献类型：
--
作者：
Sweetser,DA;Hauft,SM;Hoppe,PC;Birkenmeier,EH;Gordon,JI
大鼠肠道脂肪酸结合蛋白(I-FABP)基因在不断再生的小肠上皮中的表达存在细胞特异性和区域差异。为了研究其潜在的机制，我们将其5‘非转录结构域的一部分连接到人生长激素(HGH)基因，并通过RNA印迹、溶液杂交和免疫细胞化学技术分析了hGH报告基因在转基因小鼠中的表达。位于I-FABP转录起始点277个核苷酸内的序列足以限制hGH在肠道中的表达。虽然这些转基因小鼠十二指肠和空肠近端hGH mRNA的绝对水平与I-FABP mRNA相似，但在它们的远端小肠中，hGH mRNA的稳态浓度大约低100倍。添加I-FABP基因的-278至-1178核苷酸可将远端空肠和回肠的hGH mRNA浓度“恢复”到与小鼠I-FABP mRNA相当的水平。“短启动子”转基因小鼠的血清hGH水平比“长启动子”转基因小鼠低1000倍，这表明高效的小肠远端hGH表达需要产生更高的血清hGH浓度。HGH在绒毛相关的肠细胞和杯状细胞中的分布以及在Lieberkuhn隐窝中的表达缺失与所有转基因家系中内源性I-FABP基因产物的表达相似。然而，在短启动子转基因杂合子的小鼠中，经常可以观察到hGH阴性细胞条带从绒毛底部延伸到绒毛顶端。I-FABP没有观察到这种镶嵌染色。这些数据表明：(I)完整表达近端-远端I-FABP梯度可能需要不同的顺式作用序列，而不是重现其正常的隐窝绒毛顶端分布所需的顺式作用序列；(Ii)在位于小肠不同区域的肠细胞内，分泌蛋白的输出途径可能存在差异；(Iii)通过观察转基因沿绒毛上皮的镶嵌模式，在体内可以检测到不同的隐窝干细胞之间可能存在细微的遗传差异。
The rat intestinal fatty acid binding protein (I-FABP) gene exhibits cell-specific as well as regional differences in its expression within the continuously regenerating small intestinal epithelium. To investigate the underlying mechanisms, we linked portions of its 5' nontranscribed domain to the human growth hormone (hGH) gene and analyzed expression of the hGH reporter in transgenic mice by RNA blot, solution hybridization, and immunocytochemical techniques. Sequences located within 277 nucleotides of the start site of I-FABP transcription are sufficient to limit hGH expression to the intestine. Although the absolute levels of hGH mRNA in the duodenum and proximal jejunum of these transgenic mice were similar to those of I-FABP mRNA, steady-state hGH mRNA concentrations were approximately 100 times lower in their distal small intestine. Addition of nucleotides -278 to -1178 of the I-FABP gene "restored" hGH mRNA concentrations in the distal jejunum and ileum to levels comparable to murine I-FABP mRNA. Serum hGH levels were 1000 times lower in the "short promoter" transgenic mice compared to animals with the "long promoter" transgene, indicating that efficient distal small intestinal hGH expression is required to produce elevated hGH concentrations in serum. The distribution of hGH in villus-associated enterocytes and goblet cells and its lack of expression in the crypts of Lieberkuhn mimicked that of the endogenous I-FABP gene product in all transgenic pedigrees. However, bands of hGH-negative cells extending from the base to the tips of villi were frequently observed in mice that were heterozygous for the short promoter transgene. This mosaic staining was not observed for I-FABP. These data suggest that (i) different cis-acting sequences may be required for complete expression of proximal-distal I-FABP gradients than for recapitulation of its normal crypt-villus tip distribution; (ii) differences may exist in the export pathways of secreted proteins within enterocytes located in various regions of the small intestine; and (iii) there may be subtle genetic differences among various crypt stem cells that can be detected in vivo by observing mosaic patterns of transgene expression along the villus epithelium.