Bi L, Sarkar R, Naas T, Lawler A M, Pain J, Shumaker S L, Bedian V, Kazazian H H
Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Blood. 1996 Nov 1;88(9):3446-50.
Previously we created two strains of factor VIII-deficient mice by insertion of a neo gene into (1) the 3' end of exon 16 and (2) exon 17 of the factor VIII gene. Affected mice of both strains have no plasma factor VIII activity, yet are healthy with no spontaneous bleeding. Factor VIII-deficient females bred with affected males survive pregnancy and delivery. We used reverse transcriptase-polymerase chain reaction of liver RNA to characterize factor VIII mRNA processing. Factor VIII mRNA of the exon 16 knockout strain contains neo sequences plus 17 bp of intron 16 due to use of a cryptic donor site in intron 16. All factor VIII mRNA of the exon 17 knockout strain lacks exon 17 and neo sequences. In skipping exon 17, the intron 16 donor site or a cryptic donor site 46 bp 3' to the intron 16 donor site are used. Thus, factor VIII deficiency in exon 16 knockout mice is due to truncated protein, while in exon 17 knockout mice it is due to either truncated or partially deleted protein. After immunizing exon 16 knockout mice with human recombinant factor VIII, two monoclonal antibodies were obtained that recognize < 100 pg of mouse factor VIII light chain. Assay of cryoprecipitate from the plasma of affected mice failed to show factor VIII light chain.
此前，我们通过将新霉素基因插入（1）外显子16的3'末端和（2）凝血因子VIII基因的外显子17，创建了两种凝血因子VIII缺陷型小鼠品系。两种品系的患病小鼠均无血浆凝血因子VIII活性，但健康且无自发性出血。凝血因子VIII缺陷型雌性小鼠与患病雄性小鼠交配后可存活至怀孕和分娩。我们使用肝脏RNA的逆转录聚合酶链反应来表征凝血因子VIII mRNA的加工过程。外显子16敲除品系的凝血因子VIII mRNA由于使用了内含子16中的一个隐蔽供体位点，包含新霉素序列以及17 bp的内含子16。外显子17敲除品系的所有凝血因子VIII mRNA均缺少外显子17和新霉素序列。在跳过外显子17时，使用了内含子16供体位点或内含子16供体位点下游46 bp处的一个隐蔽供体位点。因此，外显子16敲除小鼠中的凝血因子VIII缺乏是由于蛋白质截短，而在外显子17敲除小鼠中则是由于蛋白质截短或部分缺失。用人重组凝血因子VIII免疫外显子16敲除小鼠后，获得了两种单克隆抗体，它们可识别<100 pg的小鼠凝血因子VIII轻链。对患病小鼠血浆的冷沉淀进行检测未显示凝血因子VIII轻链。
