Xu Z, Gupta V, Lei D, Holmes A, Carlson E, Gruenert D C
Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA.
Gene. 1998 Apr 28;211(1):117-23. doi: 10.1016/s0378-1119(98)00090-0.
Cystic fibrosis (CF) is a severe autosomal-linked inherited disease in humans. Transgenic CF animals play a crucial role in the study of molecular mechanisms underlying disease pathology. In the present study, CFTR mRNA expression was examined in different tissues from one CF mouse model that contains a disruption in exon 10 sequence of the CF transmembrane conductance regulator (CFTR) gene. Multiple tissue samples were collected from new-born normal (+/+), homozygous (-/-), and heterozygous (+/-) mice and compared for their CFTR mRNA expression. Total RNA samples were prepared from eight different tissues (nasal mucosa, trachea, lung, colon, intestine, pancreas, liver, gonads, and brain) and then analyzed by reverse transcription polymerase chain reaction (RT-PCR) amplification. A 329-bp fragment comprising exon 9 through exon 11 of the CFTR gene was amplified from all tissues of the normal mouse. In contrast, a 137-bp fragment was observed in tissue samples from homozygous CF mice. Both the 329-bp and 137-bp fragments were detected in samples from heterozygous mice. Direct sequencing analysis of the amplified fragments showed an exon 9-exon 11 splice junction, indicating that the entire exon 10 sequence was eliminated from homozygous CF mice. RT-PCR analysis of the 3' end of CFTR mRNA showed the presence of a 682-bp, exon 20-24 fragment in -/- and +/- mice. These results demonstrate that an alternately spliced CFTR mRNA is produced in this CF 'knock-out' mouse. A semi-quantitative comparison of the wild-type and exon 10 minus CFTR (CFTR-E10) mRNA in heterozygote animals indicated that less (but a detectable amount) mutant CFTR mRNA was present in all organs tested. There was, however, a significant reduction of CFTR-E10 mRNA in the liver and the pancreas. Since the deletion of exon 10 is in-frame, the significance of the CFTR-E10 mRNA in terms of CFTR protein and function requires further analysis.
囊性纤维化（CF）是人类一种严重的常染色体连锁遗传病。转基因CF动物在疾病病理潜在分子机制的研究中发挥着关键作用。在本研究中，在一种CF小鼠模型的不同组织中检测了囊性纤维化跨膜传导调节因子（CFTR）mRNA的表达，该模型的CFTR基因第10外显子序列存在破坏。从新生的正常（+/+）、纯合子（-/-）和杂合子（+/-）小鼠中收集多个组织样本，并比较它们的CFTR mRNA表达。从八个不同组织（鼻粘膜、气管、肺、结肠、小肠、胰腺、肝脏、性腺和脑）制备总RNA样本，然后通过逆转录聚合酶链反应（RT-PCR）扩增进行分析。从正常小鼠的所有组织中扩增出一个包含CFTR基因第9外显子至第1外显子的329 bp片段。相反，在纯合子CF小鼠的组织样本中观察到一个137 bp的片段。在杂合子小鼠的样本中检测到了329 bp和137 bp的片段。对扩增片段的直接测序分析显示存在第9外显子-第11外显子剪接连接，表明纯合子CF小鼠中整个第10外显子序列被消除。对CFTR mRNA 3'端的RT-PCR分析显示，在-/-和+/-小鼠中存在一个682 bp、第20-24外显子的片段。这些结果表明，在这种CF“敲除”小鼠中产生了一种交替剪接的CFTR mRNA。对杂合子动物中野生型和缺失第外显子10的CFTR（CFTR-E10）mRNA进行半定量比较表明，在所有测试器官中存在较少（但可检测到）的突变CFTR mRNA。然而，肝脏和胰腺中CFTR-E10 mRNA显著减少。由于第10外显子的缺失是框内缺失，CFTR-E10 mRNA在CFTR蛋白和功能方面的意义需要进一步分析。
