Preparation method and purpose of mitofilin gene knockout mice model
A gene knockout, mouse technology, applied in the field of preparation of Mitofilin heterozygous mice, can solve the problem of unclear function of Mitofilin
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Embodiment 1
[0043] The establishment of embodiment 1.Miotifilin knockout mice
[0044] In the present invention, the Mitofilin mutated ES cell line (gifted by the British Sanger Institute) is injected into the C57BL / 6J mouse blastocyst by blastocyst injection, and the embryo is transplanted into the uterus of a pseudopregnant mouse, and the Mitofilin mutant cells are integrated into the germline chimeric mice. Mitofilin heterozygous mice were obtained after mating the chimeras with wild-type mice. The specific operation steps are as follows:
[0045] a) isolating and culturing embryonic fibroblasts of mice, and treating them with mitomycin C for culturing the trophoblast of ES cells;
[0046] b) After the revived ES cells are cultured on a culture dish covered with trophoblast cells, they are injected into blastocysts of surrogate mice;
[0047] c) The injected blastocysts are placed in culture drops added with Brinster's BMOC-3, and marked for transplantation;
[0048] d) transferrin...
Embodiment 2
[0049] Example 2. Mitofilin knockout mouse homozygote dies at embryonic day 7.5-9.5
[0050] The present invention selects mice with C57BL / 6 / 129Ola heterozygous background and 129Ola background as experimental mice. Mitofilin heterozygous mice in these two backgrounds can be born normally and conform to Mendelian law. There was no significant difference between Mitofilin heterozygous mice and wild-type mice. Western Blot detected that the expression level of Mitofilin in Mitofilin heterozygous mice was lower than that of wild type, which was consistent with the lack of haploid efficiency. We performed PCR, Souther Blot and Western Blot on the progeny produced by mating Mitofilin heterozygous mice ( figure 2 ). No surviving Mitofilin homozygous mice were born in either background. Then we counted the offspring, and the proportions of wild type and heterozygote were 1 / 3 and 2 / 3 respectively (Table 1). This is consistent with the Mendelian law of homozygous embryo lethality...
Embodiment 3
[0059] Example 3. Mitofilin homozygous embryo mitochondrial ultrastructure changes
[0060] The offspring embryos mated with 8.5-day-old Mitofilin heterozygous mice were sliced, and their mitochondrial ultrastructure was observed by transmission electron microscope. Specific steps are as follows:
[0061] 1. Take Mitofilin + / - The 8.5-day embryos after mating of mice were quickly placed in pre-cooled 2.5% freshly prepared glutaraldehyde solution (0.1M PBS pH 7.4), fixed at 4°C for 2 hours, and washed 3 times with phosphate buffer .
[0062] 2. After fixing with 1% osmic acid fixative solution for 1-2 hours, dehydrate, replace and infiltrate the sample, and then embed it.
[0063] 3. Put the embedding plate into the incubator and place it at 37°C, 45°C, and 60°C for 24 hours respectively.
[0064] 4. Block trimming: Clamp the embedding block on a special holder, place it under a dissecting microscope, use a sharp blade to cut off the embedding agent on the surface to expose...
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