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Modified method for preparing genetically-modified mice by ES (embryonic stem) cell gene targeting technique

A cell gene and gene modification technology, applied in the field of biology and biological genes, can solve the problems of difficulty in obtaining complete source mice, difficulty in obtaining pre-blastocyst embryos, and death of offspring, so as to reduce the demand for equipment, low cost, and The effect of simplifying the operation process

Active Publication Date: 2017-05-31
CYAGEN BIOSCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] (1) The disadvantage of the embryonic stem cell blastocyst injection method is that generally only chimeras can be produced, and it is difficult to obtain mice completely derived from exogenous ES cells, which are not truly genetically modified mice. Select truly stable genetically modified mice (and only account for 50% of the total number of offspring) from among them. This passaging process will take at least 3 months longer, and the success rate is low (less than 1 / 3 on average);
[0010] (2) The disadvantage of tetraploid embryo ES microinjection is that the operation is more complicated, the birth rate is low, and the offspring produced when ES is derived from inbred mice often die;
[0011] (3) The disadvantage of the existing pre-blastocyst embryo ES injection method is that the efficiency of obtaining 100% ES-derived mice is low, and it is more difficult to obtain pre-blastocyst embryos than blastocysts

Method used

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  • Modified method for preparing genetically-modified mice by ES (embryonic stem) cell gene targeting technique
  • Modified method for preparing genetically-modified mice by ES (embryonic stem) cell gene targeting technique
  • Modified method for preparing genetically-modified mice by ES (embryonic stem) cell gene targeting technique

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 microinjection method comparison

[0054] 1.1 Medium required for the experiment:

[0055] The injection and embryo medium required for this experiment were prepared according to Table 2.

[0056] Table 2. Injection solution and embryo culture medium

[0057] Reagent name brand Item No. Amount added Sodium chloride NaCl Shanghai Zhanyun Chemical 7647-14-5 5.0g / L Potassium chloride KCl Shanghai Zhanyun Chemical 7447-40-7 0.4 / L Calcium Chloride CaCl 2 2H 2 o

Sigma C5080 0.5 / L Potassium dihydrogen phosphate KH 2 PO 4

Shanghai Zhanyun Chemical 7778-77-0 0.5g / L Magnesium Sulfate MgSO 4 ·7H 2 o

Shanghai Zhanyun Chemical 7487-88-9 0.2 / L Sodium bicarbonate NaHCO 3

Shanghai Zhanyun Chemical 144-55-8 20g / L β-Mercaptoethanol β-Mercaptoethanol Sigma M3148 5 mL / L Transferrin Transferrin PeproTech AF-200-02 0.02g / L glucose Sigma G7021 0.5g / L ...

Embodiment 2

[0068] Embodiment 2 microinjection effect comparison

[0069] 2.1 Medium required for the experiment:

[0070] M2 medium: purchased from SIGMA Company, the article number is M7167;

[0071] Injection of the present invention: formula as shown in table 2.

[0072] 2.2 Experimental grouping:

[0073] M group is M2 culture medium; N group is the injection solution of the present invention, and the formula is shown in Table 2.

[0074] 2.3 Experimental steps

[0075] According to the experimental operation process, the embryo acquisition and microinjection methods are all carried out according to the optimal method of the present invention, and the other step conditions are the same, the difference is only: the injection of the M group is the M2 medium; the injection of the N group is based on The injection of invention table 2.

[0076] 2.4 Experimental results: The data statistics are shown in Table 4:

[0077] Table 4 Statistical results of data

[0078]

[0079] It c...

Embodiment 3

[0080] Embodiment 3. Embryo culture medium comparative test after injection

[0081] 3.1 Medium required for the experiment:

[0082] Table 5. NB Medium Formula

[0083] Reagent name brand Item No. add ratio Knockout DMEM Gibco 10829018 70-95％ N2(100×) Gibco 17502048 0.1-10× B27(50×) Gibco 17504044 0.1-10× NEAA(100×) 注

Cyagen 10201-100 0.1-10× GlutaMAX(100×) Gibco 35050061 0.1-10× Penicillin streptomycin (1000×) Gino GNM15140 0.1-10× β-mercaptoethanol Sigma M7522 0.01-0.8mM leukemia inhibitory factor LIF Pushin 123-07 1-20ng / mL

[0084] Table 6 KSR medium formula

[0085] Reagent name brand Item No. use concentration Knockout DMEM Gibco 10829018 75-90％ KSR Gibco N10828028 5-20％ NEAA(100×) 注

Cyagen 10201-100 0.1-10× GlutaMAX(100×) Gibco 35050061 0.1-10× Penicillin streptomycin (100×) Gino GNM15140 0.1-10× β-merc...

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Abstract

The invention discloses a modified method for preparing genetically-modified mice by ES (embryonic stem) cell gene targeting technique, and is intended to improve the efficiency of acquiring 100% ES sourced genetically-modified mice by pre-blastocyst ES injection method, focusing on the improvements and optimizations for pre-blastocyst embryo acquisition method, microscopic injection method, post-microscopic embryo in-vitro culture and the like. The yield of 100% ES sourced mice is guaranteed, the cost is shortened greatly, and the cost is reduced greatly.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the field of biogene technology, in particular to an improved method for preparing genetically modified mice by ES cell gene targeting technology. Background technique [0002] ES gene targeting is a technique for site-directed integration of exogenous genes into a certain site on the genome of ES cells through homologous recombination, so as to achieve site-specific modification and modification of a gene on the chromosome. Genetically modified mice can be produced by introducing gene-targeted ES cells into embryos by microinjection. This technology is the first technology to achieve genome-specific modification, and for a long time, it is the only technology that can perform gene-specific manipulation at the genome level. Due to the long-established time of this method, high technical maturity, and no off-target effects, etc., It is still widely considered as an ideal method to mo...

Claims

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Application Information

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IPC IPC(8): C12N15/877C12N15/89A01K67/027
CPCA01K67/0271A01K2227/105A01K2267/03C12N15/8775C12N15/89
Inventor 郑敦武方明欧阳应斌俞晓峰商春华
Owner CYAGEN BIOSCI INC
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