Targeted inactivation of the coagulation factor IX gene causes hemophilia B in mice
Targeted inactivation of the coagulation factor IX gene causes hemophilia B in mice
复制标题
DOI：
10.1182/blood.v92.1.168.413k06_168_174
复制
发表时间：
1998-07-01
期刊：
影响因子：
20.3
通讯作者：
Gordon, EM
中科院分区：
文献类型：
--
作者：
Kundu, RK;Sangiorgi, F;Gordon, EM
血友病B是基因治疗的主要靶点，因为目前的治疗方法并不理想。因此，为了开发血友病B的基因治疗策略，我们建立了凝血因子IX(F.IX)缺乏的小鼠模型。通过用磷酸甘油激酶启动子驱动的新霉素抗性(neo(R))基因盒替换包含催化域的3.2kb的基因片段，创建了靶向载体。转基因的胚胎干细胞克隆产生了嵌合的雄性小鼠，并在它们的后代中观察到了灭活的F-IX基因的生殖系传播。Southern分析证实，在半合子雄性小鼠和携带者雌性小鼠中存在突变基因。从半合子小鼠分离的肝脏RNA中未检测到F-IX转录本，而携带F-IX基因的雌性小鼠的F-IX mRNA水平低于正常产仔小鼠。不出所料，患病雄鼠的凝血剂平均滴度为2.8U/dL(n=10)，而携带者雌鼠的平均凝血剂滴度为35U/dL(n=14)，而正常雌鼠的平均滴度为69U/dL(n=9)，正常雌鼠的凝血剂滴度为92U/dL(n=22)。此外，半合子小鼠的尾巴出血时间(~gt；3小时)明显延长，与正常和携带者雌性小鼠相比(15-20分钟)。在19只受影响的雄性小鼠中，有7只在剪断尾巴后死于失血，两只受影响的小鼠死于脐带出血。目前，有10只受影响的小鼠在4个月大时存活下来。除了因子IX缺陷外，携带者雌性和半合子雄性小鼠在组织学检查中没有肝脏病理，是可生育的，并以预期的孟德尔频率传播F.IX基因突变。综上所述，我们已经产生了一只F.IX基因敲除小鼠，用于评估美国血液病学会1998年的脐部血友病基因治疗策略。
Hemophilia B is a leading target for gene therapy because current therapy is not optimal. Hence, a murine model of factor IX (F. IX) deficiency was generated to develop gene therapy strategies for hemophilia B. A targeting vector was created by replacing a 3.2-kb segment of the gene encompassing the catalytic domain with a phosphoglycerokinase promoter-driven neomycin resistant (neo(r)) gene cassette. The transfected embryonic stem cell clones generated chimeric male mice, and germ line transmission of the inactivated F. IX gene was observed in their offsprings. Southern analysis confirmed the mutant genotype in hemizygous male and carrier female mice. F. IX transcripts were not detected in liver RNA isolated from hemizygous mice, and lower levels of F. IX mRNA were noted in carrier female mice when compared with those of normal litter mates. As expected, the mean F. IX coagulant titer of affected male mice was 2.8 U/dL (n = 10), while the mean F.IX titer of carrier female mice was 35 U/dL (n = 14), compared with 69 U/dL (n = 9) for the normal female mice and 92 U/dL (n = 22) for normal male and female litter mates. Further, the tail bleeding time of hemizygous mice was markedly prolonged (>3 hours) compared with those of normal and carrier female litter mates (15 to 20 minutes). Seven of 19 affected male mice died of exsanguination after tail snipping, and two affected mice died of umbilical cord bleeding. Currently, there are 10 affected mice surviving at 4 months of age. Aside from the factor IX defect, the carrier female and hemizygous male mice had no liver pathology by histologic examination, were fertile, and transmitted the F. IX gene mutation in the expected Mendelian frequency. Taken together, we have generated a F. IX knockout mouse for evaluation of navel gene therapy strategies for hemophilia B. (C) 1998 by The American Society of Hematology.