Human TCTP gene whole-body knock-down animal model with as well as preparation method and application thereof

A non-human mammal and gene technology, applied in the field of preparation of non-human mammals with whole-body knockdown of TCTP gene, can solve the problems of mouse embryo lethality and inability to obtain model animals, etc.

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

[0007] Whole-body knockout of the TCTP gene will lead to lethal mouse embryos. At present, there are no genetically engineered mice for genetic modification of this gene in China, and relevant model animals cannot be obtained in China.

Method used

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  • Human TCTP gene whole-body knock-down animal model with as well as preparation method and application thereof
  • Human TCTP gene whole-body knock-down animal model with as well as preparation method and application thereof
  • Human TCTP gene whole-body knock-down animal model with as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0183] Example 1 Construction of Targeting Vector

[0184] 1. Targeting carrier element and targeting strategy design

[0185] 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. Reporter cassette (Reporter cassette) and antibiotic resistance gene cassette (Neo gene cassette) with Frt sequence, the reporter gene cassette Reporter cassette has a constitutive splicing receptor (En2SA), which can mediate the forced splicing of mRNA, so that 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...

Embodiment 2

[0294] Example 2 Microinjection of TCTP Gene Systemic Knockdown Targeting Vector into ES Cells and Screening of On-target Clones

[0295] (1) Preparation of trophoblast cells and culture of ES cells

[0296] Mouse primary embryonic fibroblast ES (GN-M127, Gaining Biology) was taken, and treated with mitomycin C (Sangon Bioengineering (Shanghai) Co., Ltd.) with a final concentration of 10 μg / Ml after expansion for 3 hours, and finally aliquoted and frozen at -80°C. The day before thawing ES cells, pave a 0.1% gelatin-treated culture dish with a feeder layer, and culture ES cells using a culture solution supplemented with LIF (LIF concentration 1000U / Ml)

[0297] (2) Electroporation and clone screening of ES cells

[0298] 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.

[0299] ES cells were digested with trypsin and resuspended in PBS, linearized targeting...

Embodiment 3

[0344] Example 3, Blastocyst Injection of Targeted Recombinant Embryo Cells and Culture of Chimeras

[0345] Production of gene knockout animals by microinjection (Microinjection) of blastocysts, the main process includes the following steps:

[0346] 3.1 Preparation of sterile male mice and pseudopregnant female mice

[0347] Preparation of sterile male mice

[0348] 3.1.1 Preparation for anesthesia: 7-week-old C57BL / 6 male mice (National Rodent Experimental Animal Seed Center Shanghai Branch) were selected for weighing and intraperitoneally injected with 0.7% pentobarbital sodium solution.

[0349] 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).

[0350] 3.1.3 Ligation of male mice: the anesthetized C57BL / 6 male mice (National Rodent Experimental Animal Seed Center Shanghai Sub-ce...

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Abstract

The application discloses a non-human mammal model constructing kit with conditional gene knock-down of TCTP genes, a targeting vector, a constructing method of the targeting vector, a non-human mammal TCTP gene whole-body gene knock-down animal model, serums, an egg cell, a fertilized egg, an embryo, an offspring, tissues or cells of the animal model, application of products and the method in immunology research, research of immunotoxicology, immune rejection response and mechanism thereof, research of a cancer cell apoptosis mechanism, reach of a cancer target and cancer targeted therapy, safety evaluation and pharmacodynamic evaluation, production of a tumor pathologic model and preparation of drugs for preparing diseases based on a recombinase recognition system. The stably-inherited TCTP gene is constructed by using a homologous recombination technology and an embryonic stem cell technology for whole-body knock-down of a mouse, so that embryonic lethality caused by whole-body knock-down of the gene is avoided; the invention has important significance for researching occurrence and development of cancer and cancer treatment of the TCTP gene.

Description

technical field [0001] The present invention relates to the field of making whole-body gene knockdown animal models, in particular, relates to a method and application for preparing TCTP gene whole-body knockdown non-human mammals. 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 urb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/12C12N5/10A01K67/027
CPCA01K67/0271C12N5/0606C12N15/8509C12N15/907C07K14/82A01K2267/03A01K2207/12A01K2217/075A01K2227/105C12N2510/00C12N2800/30C12N2800/107
Inventor 窦科峰刘薇王琳陶开山刘奇林智斌
Owner 窦科峰
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