UOX gene knockout mouse model and construction method thereof
A technology for gene knockout mice and construction methods, which can be applied to other methods of inserting foreign genetic materials, genetic engineering, biochemical equipment and methods, etc. The effective time is short, and the possibility of complications is small, the model variability is small, and the operation is convenient.
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Embodiment 1
[0036] Embodiment 1: Selection and feeding of mice
[0037] The experimental animals were wild-type C57BL / 6 female mice (SPF grade, 3-4 weeks old); wild-type male C57BL / 6 mice (SPF grade, 6-8 weeks old), all from Changzhou Cavens Experimental Animal Co., Ltd. 【SCXK (Su) 2016-0010】. The above mice were in good health and mental condition, and no trauma or disability occurred.
[0038] The experimental animal breeding address is Changzhou Cavens Experimental Animal Co., Ltd. Breeding environment SPF grade environment, 12h alternating day and night, temperature (22±4)°C, humidity (55±10)%. The feeding time was 1 week. During the feeding period, the mice were given standard feed and clean drinking water, and the drinking water, litter and feed were changed every day.
Embodiment 2
[0039] Example 2: Obtaining of sgRNA and Cas9RNA
[0040] 1.1. Primer design
[0041] Two sgRNAs were designed at the front and back of the exon 3 functional domain of UOX gene, and PCR upstream primers UOX-sgRNA-1 and UOX-sgRNA-2, and downstream primer T7SG-RO were designed according to the sgRNA structure. The primer sequences are as follows:
[0042] UOX-sgRNA-1: 5'-ATTGCTAGCGTGGTTTGTGC-3'
[0043] UOX-sgRNA-2: 5'-GGGCTCAAGGGACCTCACACGTC-3'
[0044] T7SG-R0: 5'-AAAAGCACCGACTCGGTGCC-3'
[0045] 1.2. Preparation of transcription template
[0046] UOX-sgRNA-1, UOX-sgRNA-2 and T7SG-R0 were mixed respectively, and the sgRNA template was amplified twice by PCR after mixing with the amplification reagent and the px330-sgRNA carrier (purchased from Addgene). The reaction conditions were pre-denaturation at 98°C for 5 minutes; 35 cycles were performed according to the cycle program (98°C for 30 s, 60°C for 30 s, and 72°C for 30 s); extension at 72°C for 5 min. The reaction produ...
Embodiment 3
[0051] Example 3: Acquisition and transplantation of UOX mutant mouse embryos
[0052] Several female C57BL / 6 mice were injected with 10 IU of PMSG, followed by 10 IU of hCG the next day, and housed with male C57BL / 6 mice overnight. Euthanasia was performed on female mice with occlusion, and cumulus cell complexes were collected from oviducts. cumulus cell complex in M2 + Wash several times in hyaluronidase medium, place in KSOM medium at 35°C to 38°C, and culture in 5% CO2 incubator.
[0053] Pull, bend, and secure microinjection needles with a needle puller and a needle breaker. Inject Cas9 mRNA (100ng / μl) and sgRNA (50ng / μl) into prokaryotic embryos with a microinjection needle under a microscope, continue to culture embryos in KSOM medium for 24h to two-cell embryos, and observe embryonic cleavage and blastocysts developmental condition. Results The two-cell embryo rate was 81.7% (58 / 71). Embryos microinjected with Cas9 protein and UOX-sgRNA were transferred to 58 two-...
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