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TCTP gene tissue specific knock-down animal model, preparation method thereof and application thereof

A tissue-specific and gene-based technology, applied in the field of preparing mice with tissue-specific knockdown of the TCTP gene, can solve the problem of mouse embryo lethality

Pending Publication Date: 2018-12-28
窦科峰 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whole-body knockout of TCTP gene will lead to mouse embryo lethality, currently there is no genetically engineered mouse for genetic modification of this gene in China
As a multifunctional proto-oncogene, TCTP gene is a multifunctional proto-oncogene. To study its mechanism in various cancers, tissue-specific TCTP gene knockout engineering mice, which can avoid systemic knockout and lethal knockout of TCTP gene, are needed as a platform, which is currently unavailable in China. Mice-like products

Method used

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  • TCTP gene tissue specific knock-down animal model, preparation method thereof and application thereof
  • TCTP gene tissue specific knock-down animal model, preparation method thereof and application thereof
  • TCTP gene tissue specific knock-down animal model, preparation method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0209] Example 1 Construction of Targeting Vector

[0210] 1. Targeting carrier element and targeting strategy design

[0211] In this example, the mouse TCTP gene knockdown targeting vector adopts the design scheme of first full knockout and then conditional knockout. This design strategy is to insert a flanking band upstream of 5'loxP on the basis of conventional conditional knockout. A reporter cassette (Reporter cassette) and an antibiotic resistance gene cassette (Neo gene cassette) with Frt sequences. The reporter cassette Reporter cassette has a constitutive cleavage receptor (En2 SA), which can mediate the forced cleavage of mRNA, The Reporter cassette can be expressed under the drive of the target gene promoter, so it can be used to trace the expression of the target gene. At the same time, due to the presence of polyA in the Reporter cassette, the transcription of the target gene was terminated in advance, thereby achieving the purpose of knocking out the gene. In ...

Embodiment 2

[0314] Example 2 Microinjection of TCTP Gene Systemic Conditional Knockdown Targeting Vector into ES Cells and Screening of On-target Clones (1) Preparation of Trophoblast Cells and Culture of ES Cells

[0315] Primary mouse embryonic fibroblasts were taken and treated with mitomycin C for 3 hours after expansion. The final concentration of mitomycin C was 10 μg / Ml, and finally aliquoted and stored at -80°C. The day before thawing ES cells, spread a 0.1% gelatin-treated culture dish with a feeder layer, and culture ES cells using a culture medium supplemented with LIF (LIF concentration 1000U / Ml)

[0316] (2). Electroporation of ES cells and clone screening

[0317] The targeting vector was linearized with MluI / EcoRV and NotI enzymes before transfection. Or select two enzymes, ScaII and EcoRV, according to the targeting strategy. The linearization enzyme digestion reaction system and reaction procedure were as follows: ES cells were digested with trypsin and resuspended in P...

Embodiment 3

[0346] Example 3, Blastocyst Injection of Targeted Recombinant Embryo Cells and Culture of Chimeras

[0347] Production of gene knockout animals by microinjection (Microinjection) of blastocysts, the main process includes the following steps:

[0348] 3.1 Preparation of sterile male mice and pseudopregnant females

[0349] Preparation of sterile male mice

[0350] 3.1.1 Preparation for anesthesia: 7-week-old male mice were selected for weighing and intraperitoneally injected with 0.7% pentobarbital sodium solution.

[0351] 3.1.2 Prepare surgical instruments: 3 pairs of ophthalmic forceps, 1 pair of ophthalmic scissors, 1 pair of shearing scissors, alcohol lamp, alcohol watering can, triangular needle, suture thread, sterile filter paper (about 15cm in diameter).

[0352] 3.1.3 Ligation of male mice: Clip the hair of the abdomen of the anesthetized male mice 2cm away from the genitals, wipe and disinfect with 70% alcohol cotton, open the skin layer and muscle layer respectiv...

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Abstract

The invention discloses a TCTP gene space-time specificity conditional and tissue specific gene knock-down animal model construction kit based on a recombinase recognition system, a targeting vector and a construction method thereof of the targeting vector, a mouse TCTP gene specific gene knock-down animal model and a sperm thereof, an oocyte thereof, an oosperm thereof, an embryo thereof, a filial generation thereof, a tissue thereof or a cell thereof as well as applications of the products and methods in an immunology research, researches of immunotoxicology, immunological rejection and andmechanisms thereof, researches of cancer cell apoptosis mechanisms and cancer targets, a research of targeted cancer therapy, safety evaluation, a purpose in the pharmacodynamic evaluation aspect, making of a tumor pathological model and a preparation of a disease treating medicine. By using a homologous recombination technology and an embryonic stem cell technology, a stably genetic TCTP gene knockout mouse is constructed; and therefore, embryonic death caused by knockout of the gene is avoided; and important significance in researches of the TCTP gene in cancer occurrence, progression and treatment are realized.

Description

technical field [0001] The present invention relates to the field of making tissue-specific gene knockdown animal models, in particular, relates to a method and application for preparing TCTP gene whole-tissue-specific knockout mice. Background technique [0002] Cancer is one of the main chronic diseases affecting the health of residents in our country, ranking first among the causes of death in cities. In China, there are 4.29 million new cancer cases each year, which means that about 10,000 people are diagnosed with cancer every day, and about 7 people are diagnosed with cancer every minute. If the average life expectancy in China is 85 years, then the cumulative risk of cancer per person is as high as 36%. Worldwide, approximately 22% of new cancer cases and 27% of cancer deaths occurred in China. Lung cancer is the most common malignant tumor and the leading cause of cancer death in China and even in the world. With the further acceleration of industrialization and u...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90A01K67/027
CPCA01K67/0276C12N15/85C12N15/907A01K2267/03A01K2217/075A01K2227/105
Inventor 窦科峰刘薇陶开山王琳林智斌刘奇
Owner 窦科峰
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