Targeting vector, method for constructing F4/80-DTR transgenic mouse capable of regulating, controlling and removing macrophages through diphtheria toxin, and application for targeting vector

A technology of transgenic mice and targeting vectors, applied in receptors/cell surface antigens/cell surface determinants, other methods and applications of inserting foreign genetic materials, etc. Clearing and other issues to achieve a good clearing effect

Pending Publication Date: 2020-03-31
THE FIRST AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods rely on the phagocytic ability of macrophages. Macrophages with weak phagocytic ability cannot be cleared, and the specificity of removing macrophages is not strong enough. Some non-mo

Method used

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  • Targeting vector, method for constructing F4/80-DTR transgenic mouse capable of regulating, controlling and removing macrophages through diphtheria toxin, and application for targeting vector
  • Targeting vector, method for constructing F4/80-DTR transgenic mouse capable of regulating, controlling and removing macrophages through diphtheria toxin, and application for targeting vector
  • Targeting vector, method for constructing F4/80-DTR transgenic mouse capable of regulating, controlling and removing macrophages through diphtheria toxin, and application for targeting vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Construction of a targeting vector with a long homology arm that targets and integrates the foreign gene IRES-DTR to F4 / 80 exon 22

[0073] The mouse F4 / 80 genome sequence was retrieved and downloaded from the genome.ucsc.edu website, combined with the mouse F4 / 80 (Genbank No: NM_010130.4) sequence, to determine the position and sequence of each exon and intron. BAC Clone: ​​RP23-212F14 containing the entire F4 / 80 gene was purchased from Chieldren’hospotal Oakland Research Institute, and BAC DNA was used HiPure Plasmid Filter Maxiprep Kit (Invitrogen) was prepared for use.

[0074] PCR amplification target gene fragment F4 / 80 5'side homology arm (retrieval 5HA) and F4 / 80 3'side homology arm (retrieval 3HA): use RP23-212F14 BAC DNA as template and use forward primer F4 / 80 -180659-F(NotI)-ACCACCGCGGCCGCGACAGCCAAATATTGGCAT (SEQ ID NO.1) and reverse primer F4 / 80-180900-R(SpeI)-ACCACCACTAGTTACCTGCA AACCCCCAATAA (SEQ ID NO.2) for PCR amplification of F4 / 80 5'side Homo...

Embodiment 2

[0089] Example 2: Preparation of mouse embryonic stem (ES) cells targeted to insert IRES-DTR to F4 / 80 exon 22

[0090] Mouse embryonic fibroblasts (MEFs) culture: MEFs (ATCC) are cultured and maintained in MEF medium, passaged in time and frozen. MEF medium is DMEM medium with 10% FBS, 100U / ml penicillin streptomycin, 0.05mM 2 mercaptoethanol, 2mM L-glutamine.

[0091] Balb / c ES cell culture: MEFs cells were inactivated with 30Gray gamma rays before use as trophoblast cells, Balb / c ES cells (Merck) were inoculated on the inactivated MEFs, and cultured and maintained with ES medium. ES cells grow to 70% abundance and pass in a ratio of 1:3. ES medium is DMEM medium with 15% FBS, 100U / ml penicillin, 1mM sodium pyruvate, 0.1mM non-essential amino acids, 0.05mM 2 mercaptoethanol, 2mM L-glutamine, 1μg / L leukemia inhibitory factor (LIF).

[0092] Electrotransduction of ES cells: After the ES cells are collected by trypsinization, they are washed once with PBS, and resuspended in electro...

example 3

[0099] Example 3: Breeding F4 / 80-DTR transgenic mice with targeted insertion of IRES-DTR to F4 / 80 exon 22

[0100] Blastocyst injection of ES cells to obtain F4 / 80-DTR transgenic chimera mice: select C57BL / 6J male mice with well-developed 8 weeks of age, and C57BL / 6J female mice in a 1:2 cage, and pick the positive vaginal plug the next morning The female mouse can obtain blastocysts in the uterus after 5 days of pregnancy. The ligated KM male mouse and the normal KM female mouse were caged in a ratio of 1:2, and the female mouse with a vaginal plug and swollen and ruddy vulva was selected as a pseudo-pregnant KM female mouse. ES cells targeted to insert IRES-DTR to F4 / 80 exon 22 were injected into C57BL / 6J mouse blastocysts, and then inoculated into the uterus of pseudopregnant KM female mice, and chimeric mice were born ( F0), that is, C57-derived cells and BALB / c-derived ES cells co-exist in the same individual, and the animal's coat color is mixed with black and white.

[010...

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Abstract

The invention relates to the field of animal model construction, and specifically discloses a targeting vector, a method for constructing an F4/80-DTR transgenic mouse capable of regulating, controlling and removing macrophages through diphtheria toxin, and an application for the targeting vector. The targeting vector has a sequence as shown in SEQ ID NO. 8, and is named as an F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/80 3HA L-MCL-HSV TK vector. The invention also provides a method and application examples for cell production and cultivation of transgenic mice. According to the invention, the constructed F4/80-DTR transgenic mouse animal model for regulating, controlling and removing tissue intrinsic macrophages through the diphtheria toxin can reach the tissue intrinsic macrophage removing efficiency of 70%-90%, can well remove the tissue intrinsic macrophages, is not affected in a macrophage function when the diphtheria toxin is not used for induction, is free of side reaction when the diphtheria toxin induces to remove the macrophages, provides a brand novel principle and a method for removing the tissue intrinsic macrophages, and provides a brand novel animal model capable of inducing to remove the tissue intrinsic macrophages.

Description

Technical field [0001] The invention relates to the field of animal model construction, and mainly relates to a targeting vector and a method and application for constructing F4 / 80-DTR transgenic mice for regulating and clearing macrophages by diphtheria toxin. Background technique [0002] The mononuclear macrophage system includes premonocytes in bone marrow, monocytes in peripheral blood, and macrophages in tissues. In recent years, studies have found that most of the macrophages in tissues are inherent to the tissues and formed as early as the embryonic development stage, and self-renew and maintain homeostasis in the tissues, and have nothing to do with the monocytes in the peripheral blood after adulthood . Macrophages in different tissues can have tissue-specific functions. For example, macrophages in the brain (ie microglia) can trim synapses during development, and splenic red pulp macrophages can swallow red blood cells to recover heme Peritoneal macrophages regulate ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90A01K67/027C12N15/12
CPCC12N15/8509C12N15/907A01K67/0278C07K14/705C07K14/70596C12N2800/107C12N2800/50C12N2840/203A01K2217/072A01K2227/105A01K2267/03
Inventor 盛剑鹏汤江辉白雪莉梁廷波
Owner THE FIRST AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
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