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Method for generating model of mice in male sterility

A male sterile, mouse technology, applied in the field of transgenic technology, can solve the problems of spermatogonial stem cell apoptosis, unsatisfactory animal models, and lack of blood stem cell apoptosis.

Inactive Publication Date: 2011-01-26
SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, animal models of spermatogenesis disorders, especially male infertility models, are artificially produced mainly by injecting or feeding busulfan to mice (Busulfan, namely butylene glycol diester methanesulfonate, is an alkylating agent, clinically It is mainly used for the treatment of myeloid leukemia) drugs, which induce the apoptosis of spermatogonial stem cells. The production process of this mouse model is long and requires a large number of mice. Under the action of a light dose, a small amount of residual endogenous Sperm stem cells can restart spermatogenesis; high doses, often due to serious side effects of drugs, not only cause the apoptosis of spermatogonial stem cells, but also cause the loss of apoptosis of other embryonic stem cells including blood stem cells, accompanied by bleeding, herpes, Cramps, diarrhea, nausea, vomiting, dyspnea, reduced immunity, birth defects in pregnant mice, etc., can also cause other cancerous changes in the body, such as leukemia, which will affect the overall growth and development of mice. Work brings adverse effects, this artificially generated animal model is not satisfactory

Method used

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  • Method for generating model of mice in male sterility
  • Method for generating model of mice in male sterility
  • Method for generating model of mice in male sterility

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] PLAG1-EGFP transgenic plasmid construction

[0055] About 100 mg of mouse tumor tissue or placenta tissue was used to extract total RNA according to the instructions of Trizol reagent (Gibico Company), and reverse transcription was performed with AMV standard RT-PCR kit of TaKaRa Company. PCR primers were designed based on the GenBack U65002 sequence as a template, and the following primers were used:

[0056] Primer 1 (381): 5'-TTACGACACCATGAAACTTGAG-3' (SEQ ID NO: 1)

[0057] Primer 2 (2003): 5'-TGAATCCATGTCCCAGAATCCT-3' (SEQ ID NO: 2)

[0058] A 1623bp nucleotide product containing the complete coding frame of PLAG1 and more than 100 bases before the start codon was amplified by PCR, and cloned into the pGEM-Teasy vector (purchased from Clontech Company) to obtain the pGEM-PLAG1 plasmid.

[0059] After no mismatch was confirmed by sequencing, the fragment was excised from the pGEM-PLAG1 plasmid by EcoR1 digestion, inserted into the EcoRI site of the pCMV-EGFP vecto...

Embodiment 2

[0061] Preparation of transgenic mice

[0062] The pCMV-EGFP / PLAG1 plasmid was digested with NsiI, and a 3.8 kb DNA fragment was recovered for microinjection.

[0063] 6-7 weeks old C57BL / 6J×CBA F1 female mice were taken, and fertilized eggs were collected after superovulation. The 3.8kb DNA fragment was injected into the male pronucleus of the fertilized egg through micromanipulation, and then the injected fertilized egg was implanted into one side of the fallopian tube of the pseudopregnant mouse. After 3 weeks of pregnancy, 10 transgenic mice were obtained.

[0064] Primer

Embodiment 3

[0066] Transgene Mapping by Genome Sequencing

[0067] For the 8 transgenic mouse lines obtained in Example 2, the tails were cut to extract DNA. After using Sau3AI to digest the mouse genomic DNA, add linkers at both ends, connect with DNA ligase, use primers based on the known sequences at both ends of the pGEM-Teasy vector (purchased from Clontech Company) and primers based on the sequences of the linkers at both ends Paired for PCR amplification, the PCR amplification products were ligated and cloned into the pGEM-Teasy vector, sequenced, and the adjacent genome sequence inserted with the transgene was obtained.

[0068] Sequencing results showed that the 5′-end contiguous sequence of the EGFP / PLAG1 insert fragment was as follows:

[0069] gctcccggcc gccatggcgg ccgcgggaat tcgattcaat aggggggcgta cttggcatat 60

[0070] gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc 120

[0071] aatggaaagt ccctattggc gttactatgg gagcatacgt cattattggc gtcaatgggc 180

[00...

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Abstract

A male sterile mouse model with high genetic stability and phynotype stability is disclosed. Its preparing process features that the gene Kif18a is removed from genome or the exogenous gene is inserted in the gene Kif18a to make it be deactivated. Said model can be used to research the generation and development of sperm, the gene associated with the development of sperm, the transplantation of stem spermatogonium, reconfiguration of sperm, the migration and differentiation of reproduction cells, and the medicine for contraception.

Description

technical field [0001] The invention relates to the field of transgenic technology. More specifically, it relates to a male sterile mouse model, a method for establishing the male sterile mouse model, and uses of the male sterile mouse model. Background technique [0002] The normal maintenance of spermatogenesis not only depends on the close contact between testicular germ cells and Sertoli cells, but also depends on the normal self-proliferation and differentiation of spermatogonial stem cells (SSCs) located in the basement membrane of the seminiferous tubules. Normal animals (including humans) SSCs can not only proliferate to produce new stem cells, but also develop and differentiate into spermatogenic cells at all levels. The ratio between new stem cells and differentiated cells produced by SSCs depends on specific physiological and environmental conditions, and many genes are involved in the proliferation and differentiation of germ cells (such as Vasa, Znf, Kssk, c-KI...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K49/00
Inventor 王铸钢费俭任维华
Owner SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV
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