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Method for preparing transgenic animal

A technology of transgenic and transgenic mice, applied in the field of genetic engineering, can solve the problems of low immunogenicity, and achieve the effects of simple operation, high positive rate and time saving.

Inactive Publication Date: 2010-12-22
YANGZHOU UNIV
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Problems solved by technology

e. The transposon system can be given in the form of naked DNA, or it can be transposed by combining DNA (transposon) and transposase provided in the form of RNA / protein, so its immunogenicity is low

Method used

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  • Method for preparing transgenic animal
  • Method for preparing transgenic animal
  • Method for preparing transgenic animal

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Experimental program
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Embodiment

[0033] Example: Preparation of transgenic mice (target gene: porcine seminal plasma protein II gene regulatory region and green fluorescent protein reporter gene)

[0034]1. Construction of the transgenic expression cassette: According to the published porcine seminal plasma protein II gene sequence (AJ853849) in Genbank, primers were designed, and the Landrace pig genome was used as a template to amplify the 5' and 3' of the PSP-II gene with high-fidelity DNA polymerase The flanking regions are 4087bp and 3094bp in length, and their sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; the target DNA fragments were recovered and purified by cutting the gel, and then cloned into the TA cloning vector pMD18-T according to conventional methods, and screened positive The positive clones were cloned and identified by enzyme digestion and sequencing to obtain pMD18-T-PSPII5 and pMD18-T-PSPII3 recombinant plasmids containing the 5' and 3' flanking regions of the porcine se...

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Abstract

The invention discloses a method for preparing a transgenic animal. The method is as follows: firstly, a transgenic expression cassette which contains a target gene, namely cDNA, is constructed; secondly, the transgenic expression cassette is inserted into a sleeping beauty transposon vector and a sleeping beauty transposon expression vector which contains the transgenic expression cassette is obtained; thirdly, a sleeping beauty transposon system is used for mediated transformation and a bilateral testicle multi-spot injection method is adopted for injecting a sleeping beauty transposon expression plasmid and a sleeping beauty transposase expression plasmid into the body of a male; the male is mated with a female; after animal parturition, the polymerase chain reaction and the Southern imprinting technology are utilized to analyze and determine a transgenic electropositive individual. The method uses the 'sleeping beauty' transposon system for mediated transformation during the process for preparing the transgenic animal and adopts the bilateral testicle multi-spot injection method. The method for preparing the transgenic animal has the advantages of high positive rate, time conservation, low experimental cost, practicality, simple operation and so on.

Description

technical field [0001] The invention belongs to the field of genetic engineering. Background technique [0002] Transgenic animal refers to a type of animal that integrates a foreign gene in its genome and can express and inherit it. The transgenic system breaks the phylogenetic isolation in nature, allowing genes to flow among organisms with distant phylogeny, which can not only speed up the improvement of livestock varieties, improve the quality of animal products, but also produce rare medicinal proteins. Transgenic technology is the fourth generation of genetics after linkage analysis, somatic cell inheritance and gene cloning in the 20th century. [0003] There are many ways to prepare transgenic animals, such as microinjection, retrovirus infection, embryonic stem cell-mediated method, electrotransfer method, artificial yeast chromosome method and somatic cell nuclear transfer transgenic method. [0004] Among the above-mentioned methods for preparing transgenic anim...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/89C12N15/90A01K67/027
Inventor 宋成义高波谢飞王宵燕陈国宏孙丽亚赵芹吴晗
Owner YANGZHOU UNIV
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