基因敲除鼠的建立及其表型的初步分析
摘要
线粒体通过氧化磷酸化(OXPHOS)生成ATP,为基本生命活动提供能量。线粒体嵴上承载着OXPHOS呼吸链复合物,嵴结构的复杂程度与能量需求成正比,在能量需求高的组织,线粒体嵴也很丰富。线粒体内膜的形态随着代谢状态不同而发生变化(Mannella,2006)。因此我们推测控制嵴形态的分子具有调节线粒体OXPHOS功能。Inner Membrane Protein of Mitochondria (IMMT),又称Mitofilin,是定位在线粒体内膜上,控制嵴结构的蛋白分子(John et al.,2005),为进一步在整体动物水平探讨IMMT生理功能,有必要建立Immt敲除鼠。
我们通过囊胚注射,将Immt突变的胚胎干细胞(ES)注入到受体胚胎中,得到进入种系的嵌合体而产生Immt缺失杂合子(Immt+/-)。通过5'RACE和PCR实验验证,基因诱捕载体整合至Immt基因的第三内含子中,Immt+/-小鼠杂交后出生的346只子鼠中,没有Immt缺失纯合子(Immt-/-),只有Immt+/+和Immt+/-小鼠,其比例为1：2,结合孟德尔规律推测,Immt完全缺失鼠未出生。
我们分析了Immt+/-小鼠杂交后不同时间的胚胎,来研究Immt-/-小鼠是否存在胚胎致死及胚胎致死阶段。结果证明,Immt-/-小鼠可以顺利经过囊胚阶段并着床进入原肠胚期。同窝Immt+/-和Immt+/+小鼠已发育至9.5天胚胎(E9.5)形态,而Immt-/-小鼠还停留在E7.5天形态,明显的形体小,发育迟缓,Immt-/-小鼠没有可见的心脏结构和神经管闭合。BrdU掺入实验证明Immt-/-小鼠在E8.5天仍有增殖；TUNEL实验结果表明Immt-/-胚胎细胞在E9.5天凋亡旺盛,逐渐吸收。
透射电镜超微结构显示,E7.5、E8.5、E9.5 Immt-/-胚胎线粒体嵴紊乱,肿胀,部分空化缺失。Immt-/-胚胎酶组织化学和Immt+/-小鼠肝脏体外线粒体复合物酶活性检测,共同证明IMMT缺失导致线粒体OXPHOS电子传递链末端复合物,即细胞色素c氧化酶(COX)活性明显降低,而琥珀酸脱氢酶(SDH)和ATP酶(ATPase)活性没有观察到明显变化。Immt+/- MEF线粒体膜电位上升,活性氧(ROS)生成增多。所有这些改变都证明Immt敲除鼠有明显的OXPHOS异常。
总之,Immt敲除鼠的建立,首次为阐明Immt在哺乳动物体内功能提供了研究工具。我们通过对Immt敲除鼠的表型分析,在生物体内证明,IMMT是生命中不可缺少的蛋白分子。它的缺失会导致胚胎死于E10.5天之前,始终未发育出明显的心脏组织结构,神经发育停止。缺失IMMT会引起线粒体嵴空化缺失、紊乱,COX活性下降,OXPHOS功能受损,证明IMMT在线粒体内具有极其重要的功能。
As highly dynamic double membrane bound organelles in cells, mitochondria exhibit common structural features in general but various cristae shape due to different energy demand and metabolic states, which is derived from the infolded inner membrane where protein complexes of oxidative phosphorylation and intermediate metabolism are embedded to produce ATP for the basic life activity. Abundant and complex cristae are found in mitochondria from tissues where energy demand is high. Since the fine structure of cristae change with the metabolic states and energy demand, we hypothized that mitochondrial function was modulated by the molecules which controlled the morphology of cristae. Also the effect might relate to the genesis and development of age -related disorders. Immt (Inner Membrane Protein of Mitochondria, also called Mitofilin) locates on the inner membrane of mitochondria, characterized as a controller of the cristae shape in cells. To invest the physiological functions of Immt in mice, it is necessary to establish a line of Immt deficient mice.
Gene-Trap is a gene targeting method with high efficiency. The bottleneck of gene targeting is the microinjection and screening for the chimeric mice which can breed heterozygote. Through the microinjection, we got the germline chimeric mice. We reconfirmed the heterozygote by the recombination of PCR and 5'RACE. The results from genomic PCR characterized the inserting position of gene-trap vector is in the third intron of the Immt gene. The heterozygote did not show any exterior differences. The offsprings of crossbreeding heterozygote contained one third Immt+/+ mice and two thirds heterozygote, and no Immt-/- was found after checking 346 offspring mice. Due to the Mendelian Principle in genetics, the ratio showed that the Immt-/- was not born.
To examine the details of the lethal embryos, we check embryos at different development stages from intercrossing heterozygote. It showed that all the embryos survived through implantation but no homozygote was found at E10.5 stage. Using LacZ staing and HE staing methods, we found the Immt-/- embryos remained the E7.5 size and would not grow any more until they were absorbed after E10.5 while the heterozygote and Immt+/+ ones reached E9.5 size. The further structural analysis showed the Immt-/- could not form the cardiac structure and uncompletely closed neural tube. The TUNEL results showed that the Immt-/- exhibited more apoptosis than others at the same stage of E9.5 and reabsorbed.
After examining the mitochondrial ultrastructure by electron microscope, we found the mitochondria in Immt-/- were enlarged with disorganized and partial vacuolated cristae. And enzyme histochemical staining confirmed severe mitochondrial dysfunction with decreased cytochrome c oxidase activity while the activity of succinate dehydrogenase had no detectable change. Measurements of respiratory chain enzyme activities in the liver of Immt+/- animals revealed significant reductions in the activities of COX while no obvious change in SDH. We also found that Immt-deficient mouse embryonic fibroblasts showed increased membrane potential (ΔΨm). What's more, the production of ROS in the Immt+/- MEF revealed increased. All these results confirmed severe mitochondrial OXPHOS dysfunction in Immt deficient mice.
In summary, IMMT plays an important role in controlling the fine structure of critae and the loss of Immt causes the severe OXPHOS dysfunction and the embryonic lethality in mice. The Immt-/- embryos have smaller size, delayed neural development, and indistinct somites, also with absence of cardiac structure. All these data point to a role of IMMT in OXPHOS and emphasize the role of IMMT in life.
我们通过囊胚注射,将Immt突变的胚胎干细胞(ES)注入到受体胚胎中,得到进入种系的嵌合体而产生Immt缺失杂合子(Immt+/-)。通过5'RACE和PCR实验验证,基因诱捕载体整合至Immt基因的第三内含子中,Immt+/-小鼠杂交后出生的346只子鼠中,没有Immt缺失纯合子(Immt-/-),只有Immt+/+和Immt+/-小鼠,其比例为1：2,结合孟德尔规律推测,Immt完全缺失鼠未出生。
我们分析了Immt+/-小鼠杂交后不同时间的胚胎,来研究Immt-/-小鼠是否存在胚胎致死及胚胎致死阶段。结果证明,Immt-/-小鼠可以顺利经过囊胚阶段并着床进入原肠胚期。同窝Immt+/-和Immt+/+小鼠已发育至9.5天胚胎(E9.5)形态,而Immt-/-小鼠还停留在E7.5天形态,明显的形体小,发育迟缓,Immt-/-小鼠没有可见的心脏结构和神经管闭合。BrdU掺入实验证明Immt-/-小鼠在E8.5天仍有增殖；TUNEL实验结果表明Immt-/-胚胎细胞在E9.5天凋亡旺盛,逐渐吸收。
透射电镜超微结构显示,E7.5、E8.5、E9.5 Immt-/-胚胎线粒体嵴紊乱,肿胀,部分空化缺失。Immt-/-胚胎酶组织化学和Immt+/-小鼠肝脏体外线粒体复合物酶活性检测,共同证明IMMT缺失导致线粒体OXPHOS电子传递链末端复合物,即细胞色素c氧化酶(COX)活性明显降低,而琥珀酸脱氢酶(SDH)和ATP酶(ATPase)活性没有观察到明显变化。Immt+/- MEF线粒体膜电位上升,活性氧(ROS)生成增多。所有这些改变都证明Immt敲除鼠有明显的OXPHOS异常。
总之,Immt敲除鼠的建立,首次为阐明Immt在哺乳动物体内功能提供了研究工具。我们通过对Immt敲除鼠的表型分析,在生物体内证明,IMMT是生命中不可缺少的蛋白分子。它的缺失会导致胚胎死于E10.5天之前,始终未发育出明显的心脏组织结构,神经发育停止。缺失IMMT会引起线粒体嵴空化缺失、紊乱,COX活性下降,OXPHOS功能受损,证明IMMT在线粒体内具有极其重要的功能。
As highly dynamic double membrane bound organelles in cells, mitochondria exhibit common structural features in general but various cristae shape due to different energy demand and metabolic states, which is derived from the infolded inner membrane where protein complexes of oxidative phosphorylation and intermediate metabolism are embedded to produce ATP for the basic life activity. Abundant and complex cristae are found in mitochondria from tissues where energy demand is high. Since the fine structure of cristae change with the metabolic states and energy demand, we hypothized that mitochondrial function was modulated by the molecules which controlled the morphology of cristae. Also the effect might relate to the genesis and development of age -related disorders. Immt (Inner Membrane Protein of Mitochondria, also called Mitofilin) locates on the inner membrane of mitochondria, characterized as a controller of the cristae shape in cells. To invest the physiological functions of Immt in mice, it is necessary to establish a line of Immt deficient mice.
Gene-Trap is a gene targeting method with high efficiency. The bottleneck of gene targeting is the microinjection and screening for the chimeric mice which can breed heterozygote. Through the microinjection, we got the germline chimeric mice. We reconfirmed the heterozygote by the recombination of PCR and 5'RACE. The results from genomic PCR characterized the inserting position of gene-trap vector is in the third intron of the Immt gene. The heterozygote did not show any exterior differences. The offsprings of crossbreeding heterozygote contained one third Immt+/+ mice and two thirds heterozygote, and no Immt-/- was found after checking 346 offspring mice. Due to the Mendelian Principle in genetics, the ratio showed that the Immt-/- was not born.
To examine the details of the lethal embryos, we check embryos at different development stages from intercrossing heterozygote. It showed that all the embryos survived through implantation but no homozygote was found at E10.5 stage. Using LacZ staing and HE staing methods, we found the Immt-/- embryos remained the E7.5 size and would not grow any more until they were absorbed after E10.5 while the heterozygote and Immt+/+ ones reached E9.5 size. The further structural analysis showed the Immt-/- could not form the cardiac structure and uncompletely closed neural tube. The TUNEL results showed that the Immt-/- exhibited more apoptosis than others at the same stage of E9.5 and reabsorbed.
After examining the mitochondrial ultrastructure by electron microscope, we found the mitochondria in Immt-/- were enlarged with disorganized and partial vacuolated cristae. And enzyme histochemical staining confirmed severe mitochondrial dysfunction with decreased cytochrome c oxidase activity while the activity of succinate dehydrogenase had no detectable change. Measurements of respiratory chain enzyme activities in the liver of Immt+/- animals revealed significant reductions in the activities of COX while no obvious change in SDH. We also found that Immt-deficient mouse embryonic fibroblasts showed increased membrane potential (ΔΨm). What's more, the production of ROS in the Immt+/- MEF revealed increased. All these results confirmed severe mitochondrial OXPHOS dysfunction in Immt deficient mice.
In summary, IMMT plays an important role in controlling the fine structure of critae and the loss of Immt causes the severe OXPHOS dysfunction and the embryonic lethality in mice. The Immt-/- embryos have smaller size, delayed neural development, and indistinct somites, also with absence of cardiac structure. All these data point to a role of IMMT in OXPHOS and emphasize the role of IMMT in life.
引文
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