Expression cassette and expression vector of transgenic animal coat color report gene and application thereof

A technology of transgenic animals and gene expression cassettes, which is applied in the field of constructing gene expression vectors for the coat color of transgenic animals, which can solve the problems of high cost and difficulty in detecting large animals

Inactive Publication Date: 2017-06-20
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages are: invisible to the naked eye, blue excitation light is required for excitation, and the laboratory needs to be equipped with a fluorescent microscope or blue excitation light excitation equipment. It is difficult to detect large animals, and a larger blue excitation light excitation equipment needs to be configured, and the cost is higher.

Method used

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  • Expression cassette and expression vector of transgenic animal coat color report gene and application thereof
  • Expression cassette and expression vector of transgenic animal coat color report gene and application thereof
  • Expression cassette and expression vector of transgenic animal coat color report gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0023] Embodiment 1, the construction of coat color reporter gene expression vector pPG-TYR

[0024] 1. Construction of phEN-Pro-TYR vector

[0025] First, high-fidelity PCR was used to clone bGH polyA using the pT3-PGK-NEO plasmid as a template (primer sequence: bGHpA F: TctagaCtagagctcgctgatcagcc (SEQ ID NO.1); bGHpA R: TctagaCatatgtccccagcatgcctgctatt (SEQ ID NO.2)), the reaction system was 50l, 10μL 5×SFBuffer, 1l dNTP, 2μL 10μM bGHpAF, 2l 10μM bGHpAR, 1μL PhantaTM Super-Fidelity, 1μlpT3-PGK-NEO (5ng) as template, add ultrapure water to 50μL (high-fidelity enzyme purchased from Noviazyme company). PCR amplification program: 94°C for 40s, 55°C for 40s, 72°C for 30s, 30 cycles. PCR amplification products were detected by 1.5% agarose gel electrophoresis (such as figure 1 ). The PCR product was recovered from agarose gel, connected to a T vector (purchased from Tiangen Biochemical Technology Co., Ltd.), TA cloned, and the positive clones were screened for sequencing. Afte...

Embodiment II

[0040] Embodiment II, the verification of the transgenic mice containing coat color reporter gene expression vector preparation

[0041] 1. Preparation of transgenic mice expressing TYR gene

[0042] The mouse strain used in this experiment was FVB mice. After intraperitoneal injection of 5 international units (IU) of pregnant horse serum gonadotropin (PMSG), mature female mice were injected with 2.5-5.0 IU of human chorionic gonadotropin (hCG) about 48-54 hours later, about 12 hours Ovulation can then be induced. The female mice were caged with the male mice immediately after hCG administration. The vaginal suppositories of female mice after mating are easily visible and can be indicated by mating indicators. Fertilized eggs were collected a few hours before microinjection the next morning after cage fusion. The fallopian tubes are carefully cut open, and fertilized eggs are collected by tubal irrigation or ampullotomy.

[0043] The pLB-PG-TYR plasmid was extracted with ...

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Abstract

The invention relates to construction of an expression vector of a transgenic animal coat color report gene and application thereof. An expression cassette consists of two human TYR (tyrosinase) enhancers, a human TYR promoter, a mice TYR gene and bGH polyA which are successively connected in series; the sequence thereof is showed in SEQ ID NO. 13. The invention also discloses an expression vector comprising the expression cassette. The expression vector is transferred into a host animal genome with white coat color, and melanin sedimentation is generated after transgene expression. The coat color transformation can be directly observed with naked eye at the parts of skin, hair and the like of the host animal, and target gene expression situation is directly reflected. The vector constructed by the invention is applied to prepare transgenic mouse with mice as host animals, transgenic offspring is successfully obtained, and the coat color transformation can be observed, a black mouse is obtained, the vector is proved to be applied as a report gene.

Description

technical field [0001] The invention relates to the construction and application of a transgenic animal coat color reporter gene expression vector. Background technique [0002] During the preparation of transgenic animals, reporter genes are often used to "report" the expression of the target gene. Commonly used reporter genes include chloramphenicol acetyltransferase gene, β-lactosidase gene, dihydrofolate reductase gene, fluorescent protein gene, etc. Among them, the commonly used reporter gene for live detection of transgenic animals is mainly fluorescent protein gene. The fluorescent protein family is a homologous protein with a relative molecular mass of 20-30kD found in hydrozoans and corals, including green, red, yellow and cyan fluorescent proteins. Green fluorescent protein (green fluorescent protein, GFP) is one of the most widely used, and was originally isolated from Victoria luminous jellyfish (Aequorea victoria). Osamu Shimomura, Martin Salfi and Qian Yongji...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027
CPCA01K67/027A01K2267/01C07K14/61C12N15/85C12N2800/107C12N2830/15
Inventor 高波沈丹宋成义陈伟陈才王赛赛王伟张丽王宵燕
Owner YANGZHOU UNIV
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