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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

Inactive Publication Date: 2010-12-01
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, what role does iASPPsv play in the self-renewal and directed differentiation of hematopoietic stem and progenitor cells, and the transformation of normal hematopoietic stem and progenitor cells into tumor stem cells, whether it will cause tumors in mice, and if tumors occur, whether these tumor cells Originated from stem progenitor cells, it is not clear

Method used

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  • Specificity overexpression mouse model of transduction iASPPsv cancer gene in hemopoietic system and preparation method and use thereof
  • Specificity overexpression mouse model of transduction iASPPsv cancer gene in hemopoietic system and preparation method and use thereof
  • Specificity overexpression mouse model of transduction iASPPsv cancer gene in hemopoietic system and preparation method and use thereof

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Experimental program
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Effect test

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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Abstract

The invention discloses a specificity overexpression mouse model of a transduction iASPPsv cancer gene in a hemopoietic system and a preparation method and the use thereof. The method comprises the following steps of: establishing an hM HS21 / 45vav-iASPPsv plasmid, removing a prokaryotic expression vector part after the plasmid is subjected to the Hind III enzyme digestion, and standing the product for low-voltage electrophoresis over night after the linearization followed by glue cutting, recovery, purification and nucleic acid concentration regulation. By the method of microinjection, the transgenic vector, hM HS21 / 45vav-iASPPsv, is injected to a zygote of a C57 mouse, and the zygote is implanted in a uterus of a pseudopregnancy mouse. 19 to 21 days after the operation, a new mouse is born and subjected to ear cutting, numbering and tail cutting after 3 weeks. DNA is extracted from the tail to detect the integration of the target gene, iASPPsv, by using PCR. The specificity overexpression mouse model of the transduction iASPPsv cancer gene in hemopoietic system is obtained by using molecular biology, experimental zoology and the like to successfully establish an experimental method for detecting the amplification, oriented differentiation, migration, apoptosis and the like of hematopoietic stem / progenitor cells and lay a foundation for the mechanism research and targeted therapy for further explaining the transformation of normal hematopoietic stem / progenitor cells to tumor cells.

Description

technical field [0001] The invention relates to a mouse model, in particular to a mouse model transduced with iASPPsv oncogene specifically and highly expressed in the hematopoietic system, its preparation method and application. Background technique [0002] At present, malignant tumors have become one of the most serious diseases threatening human health. With the development of social economy, the incidence of tumor is on the rise. Therefore, research on the prevention, pathogenesis, diagnosis, treatment and prognosis of various tumors has become an urgent problem to be solved. More and more studies have found that the occurrence of tumors may originate from the original tumor stem cells, and the tumor stem cells may originate from abnormally regulated normal stem cells. The translational research between the two has important implications for the prevention and pathogenesis of various tumors The related research on diagnosis, treatment and prognosis provides new ideas ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63A01K67/027A61K49/00
Inventor 王建祥彭磊文王敏饶青刘家卓邢海燕田征
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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