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Producing method of transgenic mouse containing anthropogenic proto-oncogene c-Ha-ras and application thereof

A c-ha-ras, proto-oncogene technology, applied in other methods of inserting foreign genetic materials, genetic engineering, plant genetic improvement, etc., can solve the problems of tumor confusion, unfavorable model animals, etc., and achieve the effect of fast growth

Active Publication Date: 2009-09-16
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Spontaneous tumors are unfavorable for the application of model animals in safety evaluation, because spontaneous tumors will be confused with tumors induced by test substances with carcinogenic risks

Method used

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  • Producing method of transgenic mouse containing anthropogenic proto-oncogene c-Ha-ras and application thereof
  • Producing method of transgenic mouse containing anthropogenic proto-oncogene c-Ha-ras and application thereof
  • Producing method of transgenic mouse containing anthropogenic proto-oncogene c-Ha-ras and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Isolation of human proto-oncogene c-Ha-ras DNA

[0037] 1. Structure of human proto-oncogene c-Ha-ras DNA

[0038] The invention relates to a method for making a rodent transgenic animal whose genome is integrated with human-sourced proto-oncogene c-Ha-ras DNA. The transgene (Transgene) used in the present invention is c-Ha-ras gene with a full length of 6.5kb, containing 4 exons, and containing the promoter, regulatory sequence and polyA signal sequence of the gene itself. It comes from normal human blood tissue and is 0.4kb shorter than the transgene used in previous literature reports ( figure 1 ), whose full-length sequence can be found in figure 2 .

[0039] 2. Isolation of human proto-oncogene c-Ha-ras DNA

[0040] In previous literature reports, the transgenic c-Ha-ras DNA was obtained by constructing a library (Sekiya T et al, 1984; 1985), and the starting DNA of the isolated gene came from a Japanese patient with malignant melanoma. Because the...

Embodiment 2

[0042] Example 2: Introduction of human proto-oncogene c-Ha-ras DNA

[0043] As known to those skilled in the art, the process of producing transgenic animals by microinjection (Microinjection) includes the following multiple steps: (1) preparing sterile male mice and pseudopregnant female mice; (2) superovulation; (3) harvesting fertilized eggs (4) preparation of target DNA; (5) introduction of target DNA into fertilized eggs; (6) transplantation of fertilized eggs; (7) detection of target DNA integration in Founder of the first built mouse; mouse strain. In the present invention, the microinjection methods are all carried out according to standard methods, so further descriptions are omitted. However, the present invention has established a suitable method for injecting the DNA solution into the microinjection needle during the implementation process. This method can complete the filling of the DNA solution in about 2 minutes, which is very fast. This technology has greatl...

Embodiment 3

[0044] Example 3: Selection of transgenic animals

[0045] Any suitable rodent can be used as a recipient animal for producing the "transgenic animal" of the present invention. Obviously, "transgenic animals" also include newborns, young offspring, mature animals and their embryos produced by transgenic animals; and its embryos. Examples of transgenic animals and their progeny include mice, rats, guinea pigs, etc., preferably rodents are mice, such as C57 mice. This is a mature inbred line with primer C57, the genetic background is clear and stable, and its fertilized eggs are suitable for microinjection, so it is relatively easy to make model animals.

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Abstract

The invention provides a producing method of a transgenic mouse integrated with anthropogenic proto-oncogene c-Ha-ras DNA in genomes. DNA transferred contains four coding exons, introns and regulatory sequences of human c-Ha-ras gene. The regulatory sequences are preferentially selected from the promoter region of the human c-Ha-ras gene and a poly A tailing signal sequence. The transgenic mouse,filial generation, embryo, organ and / or derived cells produced with the method can be used for canceration mechanism study, carcinogen or cancer inhibitor screening research and pre-clinical safety evaluation of drugs.

Description

technical field [0001] The invention relates to a method for making a cancer transgenic animal model; the use of the animal model in the study of carcinogenesis mechanisms of various cancers, the screening study of carcinogens or tumor suppressors, and the preclinical safety evaluation research of drugs; Derived cells, tissues, organs and embryos and their use in the above fields. Background technique [0002] Since the birth of the first transgenic mouse in the early 1980s, many gene transfer methods have been established and developed, including microinjection, retrovirus-mediated gene transfer, embryonic stem cell technology, nuclear transfer technology, gene knockout (Knock-in) technology and RNA interference, as well as sperm carriers, etc., and thus also produced many patents (US patent: US6576811B1, 2003). [0003] Among them, because of its high stability and success rate, the microinjection method has become one of the most commonly used methods for genetic modific...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N5/10C12N15/89C12Q1/04A01K67/027
Inventor 范昌发岳秉飞王军志李波左琴胡培丽
Owner NAT INST FOR FOOD & DRUG CONTROL
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