Specificity overexpression mouse model of transduction iASPPsv cancer gene in hemopoietic system and preparation method and use thereof
A hematopoietic system and mouse model technology, applied in botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of tumor occurrence and unclearness in mice, and achieve the effect of strong anti-apoptosis ability
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Embodiment 1
[0042] Example 1 Construction and purification of the transgenic vector hM HS21 / 45vav-iASPPsv carrying iASPPsv
[0043] Using high-fidelity PrimeSTAR HotStar’ DNA polymerase, PCR amplifies iASPPsv from pcDNA3.0-FLAG-iASPPsv, introduces the required Not I and Sfi I restriction sites, and the upstream and downstream primers are 5’-GCA respectively GGC CCGTACGGCC ATGTGCTGGTTG-3' (the underline is the introduction of Sfi I restriction site) and 5'-CAGC GCGGCCGC CTAGACTTTACTCCTTTG-3' (the underline is the introduction of the Not I restriction site), the reaction conditions are 98°C pre-denaturation for 1min, 98°C for 10s, 68°C for 4min, 27 cycles and then stored at 4°C, the length of the amplified fragment is 1245bp, PCR amplification After adding A tail, electrophoresis quantification ( figure 1 ) and gel recovery and purification, ligated into the pMD18-T vector, transformed into E.coli DH5α bacterial strain, carried out blue and white spot screening in LB plates, picked po...
Embodiment 2
[0044] Example 2 Establishment of iASPPsv transgenic animal model and identification of phenotype
[0045] After hM HS21 / 45vav-iASPPsv is purified, it can be integrated into the eukaryotic animal genome, such as the C57BL / 6J mouse genome, by means of microinjection, and the steps are briefly described as follows:
[0046] 1) Prepare pseudopregnant female mice (foster mothers): select several fertile female mice of the appropriate age over 2 months old as recipient mice, mate with sterilized male mice after vasectomy, and stimulate the female mice to undergo a series of pregnancy changes The obtained pseudopregnant mother mice were used as the adoptive mothers after the fertilized eggs were transgenic, and the recipient mice were observed before 9:00 in the morning of the next day, and those with sperm plugs were taken out for isolation.
[0047] 2) Preparation of fertilized eggs: select 7-8 week old female mice and fertile female mice, inject horse serum PMSG (10 IU) intraperi...
Embodiment 3
[0055] Example 3 PCR screening and identification of iASPPsv progeny positive mice and establishment of mouse model
[0056] Microinject the target nucleic acid into fertilized eggs to develop and breed mice, cut out the tail of about 1 cm from the 3-week-old mice after birth, extract genomic DNA (extract genomic DNA by phenol-chloroform-isoamyl alcohol method), and detect exogenous target by PCR Gene integration status. A total of 5 iASPPsv positive transgenic mice were identified, numbered as I-0, II-0, III-0, IV-0, V-0 ( Figure 6 ). Mate the first mouse that integrated the exogenous target gene with 6-8 week-old normal wild-type C57BL / 6J mice, pass them down, extract the tail genomic DNA of the offspring, identify the integration of the exogenous gene carried by the offspring, and detect the upstream primers The downstream primers are 5'-CTGTCCTCTGATGGGCTCTTG-3' and 5'-CTCGGGTCGTTCATCTCCTTC-3' respectively, the reaction conditions are pre-denaturation at 94°C for 5 minut...
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