CN106755113B

CN106755113B - Improved method for preparing genetically modified mouse by ES cell gene targeting technology

Info

Publication number: CN106755113B
Application number: CN201611242642.8A
Authority: CN
Inventors: 郑敦武; 方明; 欧阳应斌; 俞晓峰; 商春华
Original assignee: Cyagen Mode Biology Research Center Taicang Co ltd; Saiye Suzhou Biotechnology Co ltd
Current assignee: Cyagen Biosciences Inc Suzhou
Priority date: 2016-12-29
Filing date: 2016-12-29
Publication date: 2020-06-19
Anticipated expiration: 2036-12-29
Also published as: CN106755113A

Abstract

The invention discloses an improved method for preparing a gene modified mouse by an ES cell gene targeting technology. The invention aims to improve the efficiency of acquiring 100% ES-derived genetically modified mice by a pregerminate embryo ES injection method, and focuses on improvement and optimization in the aspects of a pregerminate embryo acquisition method, a microinjection method, in-vitro culture of microinjected embryos and the like. The invention ensures the yield of the mice with 100 percent of ES source, and greatly shortens the period and the cost.

Description

Improved method for preparing genetically modified mouse by ES cell gene targeting technology

Technical Field

The invention relates to the technical field of biology, in particular to the technical field of biological genes, and specifically relates to an improved method for preparing a gene modified mouse by an ES cell gene targeting technology.

Background

ES gene targeting is a technique for site-directed modification of a certain gene on a chromosome by integrating an exogenous gene into a certain site on the genome of ES cells through homologous recombination. Genetically modified mice can be produced by microinjecting the targeted ES cells into embryos. The technology is the earliest technology for realizing the site-specific modification of the genome, is the only technology capable of carrying out the site-specific operation of the genome at the genome level for a long time, and is still widely considered as an ideal method for modifying and transforming the genetic material of an organism at present due to the advantages of long establishment time, high technical maturity, no off-target effect and the like. However, in the present day that various new gene modification technologies are continuously appeared, compared with newly-born nuclease gene modification technologies such as CRISPR/Cas9 and the like, the preparation period of a gene modified mouse is the biggest defect of the technology.

The ES gene targeting technology for preparing the gene modified mouse can be divided into two stages of ES gene targeting and ES embryo microinjection for obtaining the gene modified mouse (hereinafter referred to as ES microinjection). The long period is mainly expressed in the ES microinjection stage, because the traditional ES microinjection is usually a chimera (only part of cells are derived from the genetically modified ES cells) and is not a truly genetically modified mouse, and a generation of new generations of cells are needed to select the truly genetically modified mouse which can be stably inherited from the offspring, and the period is at least increased by 3 months. Although ES microinjection has been developed over decades to produce a series of different methods that can produce not only chimeric progeny, but also progeny derived entirely from exogenous ES cells, the existing methods all suffer from more or less different problems.

The currently used ES microinjection methods mainly include the following methods:

(1) embryonic stem cell blastocyst injection (conventional ES microinjection): the history of more than 30 years from the 80 s of the last century to date is the main method for constructing knockout mice for a long time;

(2) tetraploid embryo ES microinjection: the method can obtain 100% of offspring mice from exogenous ES cells;

(3) ES microinjection method of pregerminate embryos: this method has the chance of obtaining 100% of progeny from exogenous ES. Since it is simpler than the conventional tetraploid embryo ES microinjection method and the offspring obtained when ES is derived from inbred mice are also not easily lethal, the method has received much attention as soon as it is born and is continuously optimized.

Although the methods have various characteristics, the methods have the following defects:

(1) the embryo stem cell blastocyst injection method has the defects that generally only chimera can be generated, mice completely derived from exogenous ES cells are difficult to obtain, the method is not a truly genetically modified mouse, a generation of new generations needs to be propagated to select the truly genetically modified mouse capable of stably inheriting from offspring (and the generation only accounts for 50% of the total number of the offspring at most), the passage process takes at least 3 months, and the success rate is low (the average is less than 1/3);

(2) the disadvantage of the tetraploid embryo ES microinjection method is that the operation is complicated, the birth rate is low, and the offspring generated when the ES comes from inbred mice is dead;

(3) the existing pre-blastocyst embryo ES injection method has the defects that the efficiency of obtaining mice with 100 percent of ES sources is lower, and the pre-blastocyst embryo obtaining difficulty is higher than that of blastocysts.

Disclosure of Invention

The invention aims to improve the efficiency of acquiring 100 percent of ES-derived genetically modified mice by a pregerminate embryo ES injection method, and focuses on improvement and optimization in the aspects of a pregerminate embryo acquisition method, a microinjection method, in-vitro culture of microinjected embryos and the like. The invention can ensure the yield of 100 percent of ES-derived mice, greatly shorten the period and reduce the cost.

The invention provides an improved method for preparing a gene modified mouse by an ES cell gene targeting technology, which comprises the following steps:

(1) providing a 2-cell embryo of a mouse,

(2) washing the 2-cell embryo several times with 5% CO at 37 deg.C₂Culturing for 12-24 hours;

(3) transferring the embryos cultured in the step (2) and the prepared ES cells into liquid drops in the same injection dish, and placing the injection dish under a microscope for microinjection; continuously sucking a plurality of ES cells into an injection needle by using the injection needle with an inclined opening under a high power microscope, selecting embryos with better shapes, inserting the needle head of the injection needle into the blastomere gap of the embryos, pushing the injector, pushing the ES cells in the injection needle into the embryos, and finally pulling out the needle;

(4) transferring the injected embryo to modified embryo culture medium, culturing at 37 deg.C and 5% CO₂Culturing for 24 hours under the condition;

(5) selecting the well-developed embryo in the step (4), and transplanting the well-developed embryo into 2.5d of uterus of a surrogate mouse;

(6) when the surrogate pregnant mice lay a young F0, the born mice are counted and identified.

Preferably, in step (2), the 2-cell embryo is washed in M2 culture drop for several times, then the 2-cell is transferred to a micro-droplet culture dish, and then after washing 2-cell embryo with well-balanced M16 culture solution for several times, finally the 2-cell embryo is placed in a clean drop.

Preferably, the microinjection of step (3) specifically comprises the following steps:

s1, selecting a single ES cell under a high power microscope, continuously sucking a plurality of cells into an injection needle by using the injection needle with an inclined opening, selecting an embryo with a better form, blowing and sucking the embryo by using a fixed needle, and rotating the embryo until a larger gap is adjusted in the direction of 3 points. Fixing the embryo by a fixing needle under negative pressure, adjusting a microscope to determine the edge of the embryo, and ensuring firm fixation of the embryo;

s2, arranging the tip of the injection needle and the center point of the embryo on the same focusing plane, and lightly contacting the surface of the embryo by using the tip of the injection needle;

s3, inserting a needle head of an injection needle into a blastomere gap of the embryo, slowly pushing an oil pressure injector, pushing the ES cells in the injection needle into the embryo, and injecting 6-8 ES cells into each embryo;

s4, after injection, the injection needle is slowly pulled out.

Preferably, the liquid drop in the injection dish in the step (3) is an injection solution, and the modified embryo culture medium in the step (4); they are identical in composition and consist of:

(ii) a Wherein the NEAA (100X) comprises the following components:

composition (I)	Concentration (g/100mL)
		Proline	0.01-0.1
Alanine	0.01-0.1
		Serine	0.01-0.1
Glycine	0.01-0.1
		Asparagine	0.05-0.5
Aspartic acid	0.05-0.5
		Glutamic acid	0.05-0.5

(ii) a Wherein the small molecular substance is amino aromatic hydrocarbons composed of C, H, F, N, O, S chemical components, and the range of the small molecular substance is C (11-25) H (9-30) F (2-8) N (1-10) O (2-8) S (1-5).

Preferably, the small molecule substance is selected from two or more of XAV939, PD184352, A0-6301, PD184352, AZD6244, CT99021, GSK1120212 and PS-341.

(ii) a Wherein the NEAA (100X) comprises the following components:

composition (I)	Concentration (g/100mL)
		Proline (L-Proline)	0.05-0.1
Alanine (L-Alanine)	0.05-0.1
		Serine (L-Serine)	0.05-0.1
Glycine (Glycine)	0.05-0.1
		Asparagine (Asparagine monohydrate)	0.05-0.4
Aspartic acid (Aspartic acid)	0.05-0.4
		Glutamic acid (Glutamic acid)	0.05-0.4

Among the components of the embryo culture medium and the injection, NEAA (100 x) is expressed as a mother solution with the NEAA being 100 x, namely the maximum dilution multiple of the NEAA is 100 times when the NEAA is applied; the maximum addition amount of the compound in the formula is 0.1-10 x, namely, the NEAA is added into the culture reagent with the total volume of 100ml to be 0.1-10 ml.

The micro-injection needle used in the invention is self-drawn, a thin-wall capillary with the inner diameter of 1mm is drawn into a slender pointed cone shape by using a P-97 needle drawing instrument of SUTTER company, the head of the pointed cone is cut off (the fracture is ensured to be neat) at the position of 15-20 mu m of the inner diameter of the capillary by using a needle forging instrument, and the fracture surface at the fracture is ground into an angle of 45 degrees by using a needle grinding instrument for standby.

At present, the most used technology for preparing the genetically modified animal model is a blastocyst ES microinjection technology, and because the technology obtains chimera which is not a truly genetically modified mouse, the technology needs to propagate one generation again to select the truly genetically modified mouse which can be stably inherited from the offspring (and the generation only accounts for 50 percent of the total number of the offspring at most), the passage process takes at least 3 months, and the success rate is low.

The microinjection technology of the invention is not equal to the efficiency of the traditional method in terms of the birth rate of mice and the ratio of ES-derived mice, and can also directly produce 100 percent of ES-derived mice in large quantity; saving time of at least 3 months; the operation process is simplified and the success rate is increased because an intermediate link is reduced. Therefore, the technology has the advantages of time saving, low cost and high efficiency, and is a breakthrough improvement on the prior art. The improved ES gene targeting technology is close to the latest nuclease gene modification technologies such as CRISPR/Cas9 and the like in the preparation efficiency of the gene modified mice, and can greatly relieve the impact on the biomedical industry in China possibly generated after the CRISPR/Cas9 gene modification technology patent takes effect in the field of gene modification.

According to the above technical solution, it can be seen that the differences between the present invention and the prior art are shown in table 1,

table 1 comparison of the invention with the prior art:

thus, the advantages of the invention over the prior art can be derived:

1. compared with the traditional method, the embryo acquisition method has the advantages that: the difficulty of obtaining the embryo is reduced, and the yield of the embryo at a specific development stage is improved.

2. Compared with the traditional method, the microinjection method of the invention has the advantages that: the requirement on equipment is reduced, the method can be realized only by common microinjection equipment, and expensive laser membrane rupture instruments and piezoelectric membrane rupture instruments do not need to be purchased.

3. Compared with the traditional method, the formula of the injection has the advantages that: the proportion of 100% ES-derived mice is greatly improved, so that the high proportion of 100% ES-derived mice can be obtained by injecting the embryo before the cyst embryo by using an oblique mouth needle.

4. Compared with the traditional method, the culture method after injection has the advantages that: greatly improves the yield of 100 percent of ES-derived mice, and makes it possible to completely replace the traditional blastocyst ES injection method by the blastocyst ES injection method.

Detailed Description

The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes and are not intended to limit the scope of the present invention. The conditions used in the examples may be further adjusted according to the conditions of the particular manufacturer, and the conditions not specified are generally the conditions in routine experiments.

EXAMPLE 1 microinjection method comparison

1.1 culture media required for the experiment:

the injection and embryo culture medium required for this experiment were prepared as shown in Table 2.

TABLE 2 injection and embryo culture media

Name of reagent	Brand	Goods number	Adding amount of
				Sodium chloride NaCl	Shanghai spreading and cloud chemical industry	7647-14-5	5.0g/L
KCl	Shanghai spreading and cloud chemical industry	7447-40-7	0.4/L
				Calcium chloride CaCl₂·2H₂O	Sigma	C5080	0.5/L
Potassium dihydrogen phosphate KH₂PO₄	Shanghai spreading and cloud chemical industry	7778-77-0	0.5g/L
				Magnesium sulfate MgSO₄·7H₂O	Shanghai spreading and cloud chemical industry	7487-88-9	0.2/L
Sodium bicarbonate NaHCO₃	Shanghai spreading and cloud chemical industry	144-55-8	20g/L
				β -Mercaptoethanol β -Mercaptoethanol	Sigma	M3148	5 mL/L
Transferrin	PeproTech	AF-200-02	0.02g/L
				Glucose	Sigma	G7021	0.5g/L
Sodium lactate	Sigma	L7022	2.0 mL/L
				Calcium lactate	Sigma	l2000	0.5g/L
Pyruvic acid sodium salt	Sigma	P5280	0.05g/L
				Ethylenediaminetetraacetic acid sodium salt	Sigma	E1644	0.05g/L
Glutamine	Sigma	49419	0.1g/L
				Bovine serum albumin	Amresco	0332	0.05g/L
Fetal bovine serum	BI	04-002-1A	7％
				HEPES	Sigma	H7523	3.5g/L
NEAA(100×)	Cyagen	10201-100	5×
				Small molecule substances			0.25g/L

(ii) a Wherein the NEAA (100X) comprises the following components:

composition (I)	Concentration (mg/L)
		Proline (L-Proline)	1150
Alanine (L-Alanine)	890
		Serine (L-Serine)	1050
Glycine (Glycine)	750
		Asparagine (Asparagine monohydrate)	1320
Aspartic acid (Aspartic acid)	1330
		Glutamic acid (Glutamic acid)	1470

(ii) a Wherein the small molecule substance is selected from two or more of XAV939, PD184352, A0-6301, PD184352, AZD6244, CT99021, GSK1120212 and PS-341.

1.2 experimental grouping:

the X1 group is piezoelectric rupture of membranes microinjection; x2 group is bevel needle microinjection

1.3 Experimental procedures and results

The method is divided into 2 groups according to different microinjection methods, wherein an X1 group uses a piezoelectric membrane rupture instrument for assisting micromanipulation, an X2 group uses an oblique needle for micromanipulation, other experimental operation steps and conditions are the same, the other experimental operation steps and conditions are all carried out according to the optimal method of the experimental process, and the statistical data of each group are as follows:

table 3 statistical data results

From the above table, it can be seen that the main data are better results obtained with the bevel needle microinjection.

EXAMPLE 2 comparison of Effect of microinjection solution

2.1 culture media required for the experiment:

m2 medium: purchased from SIGMA, inc, under item number M7167;

the injection of the invention: the formulation is shown in table 2.

2.2 experimental grouping:

group M is M2 medium; group N is the injection of the invention, and the formula is shown in Table 2.

2.3 Experimental procedures

According to the experimental operation flow, the embryo acquisition and microinjection methods are carried out according to the optimal method of the scheme of the invention, and other steps are the same in conditions, and only the differences are as follows: the injection of group M is M2 culture medium; the injections in group N are the injections in Table 2 of the present invention.

2.4 Experimental results: data statistics are shown in table 4:

TABLE 4 data statistics

As can be seen from the results, the injection of the present invention was produced at a rate of approximately 20% higher than that of 100% ES-derived mice cultured in M2 medium.

Example 3 embryo Medium comparison test after injection

3.1 culture media required for the experiment:

TABLE 5 NB Medium formulation

Name of reagent	Brand	Goods number	Proportion of addition
				Knockout DMEM	Gibco	10829018	70-95％
N2(100×)	Gibco	17502048	0.1-10×
				B27(50×)	Gibco	17504044	0.1-10×
NEAA(100×)^{Note that}	Cyagen	10201-100	0.1-10×
				GlutaMAX(100×)	Gibco	35050061	0.1-10×
Penicillin streptomycin (1000X)	Jinuo	GNM15140	0.1-10×
				β -mercaptoethanol	Sigma	M7522	0.01-0.8mM
Leukemia inhibitory factor LIF	Puxin	123-07	1-20ng/mL

TABLE 6 KSR Medium formulation

Name of reagent	Brand	Goods number	Concentration of use
				Knockout DMEM	Gibco	10829018	75-90％
KSR	Gibco	N10828028	5-20％
				NEAA(100×)^{Note that}	Cyagen	10201-100	0.1-10×
GlutaMAX(100×)	Gibco	35050061	0.1-10×
				Penicillin streptomycin (100X)	Jinuo	GNM15140	0.1-10×
β -mercaptoethanol	Sigma	M7522	0.01-0.8mM
				Leukemia inhibitory factor LIF	Puxin	123-07	1-20ng/mL

Note: the NEAA (100X) composition was as follows:

M16 medium: purchased from SIGMA corporation under the product number M7292;

KSOM medium: purchased from Millipore under the trade designation MR-106-D;

the culture medium of the invention: the compositions were prepared according to table 2.

3.2 Experimental groups:

group A: not culturing; group B: m16 medium; group C: KSOM medium; group D: KSR medium; group E: NB medium; and F group: KSOM + 4% NB; group G: the invention relates to a culture medium.

3.3 Experimental procedures and results

According to the experimental operation flow, the embryo acquisition method and the microinjection method are carried out according to the optimal method of the scheme, the conditions of other steps are the same, the difference is only that the embryo culture media of each group are different, the experiment is carried out according to the groups, and finally the statistical data of each group are as follows:

table 7 statistical data results

From the above table, it can be seen that the number of 100% ES-derived mice obtained is very small or even none if the injected embryos are not subjected to transient culture; compared with the existing culture method after injection, the method has obvious difference, and the birth rate of 100 percent of ES-derived mice is improved by 10 percent.

Example 4 comparison of the efficiency of micromanipulation of the embryos of the invention with conventional

4.1 Experimental groups:

group Y1: microinjection methods of the present invention; group Y2: traditional blastocyst ES microinjection

4.2 Experimental procedures and results

The experiment was performed in an optimal manner for the overall procedure, and conventional blastocyst ES microinjection was performed in a manner well known to those skilled in the art. When the surrogate mouse was born, the born mouse was counted and identified (F0). According to the difference that embryo donor mice are white body hair and ES source mice are black body hair, F0 mice are divided into three types according to hair color: white mice (embryonic donor-derived mice), black-and-white mottled mice (chimera mice), and pure black mice (100% ES-derived mice) were counted, respectively. The results are shown in Table 11.

Table 8 statistical data results

Because the traditional blastocyst ES microinjection method does not have 100 percent of ES-derived mice in the F0 generation, only 80 percent of chimeric mice can be used for comparison, the chimeras cannot be stably inherited, if a real gene-modified mouse is obtained, the mice must be mated again for one generation, the time of 3-4 months is wasted, the production cost and the human resource are wasted, and the success rate is not high.

The above examples are only for illustrating the technical idea and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the content of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

An improved method for preparing a genetically modified mouse by an ES cell gene targeting technology, which comprises the following steps:

(1) providing a 2-cell embryo of a mouse,

(2) washing the 2-cell embryo several times at 37 deg.C with 5% CO₂Culturing for 12-24 hours;

(3) transferring the embryos cultured in the step (2) and the prepared ES cells into liquid drops in the same injection dish, and placing the injection dish under a microscope for microinjection; continuously sucking a plurality of ES cells into an injection needle by using the injection needle with an inclined opening under a high power microscope, selecting embryos with better shapes, inserting the needle head of the injection needle into the blastomere gap of the embryos, pushing the injector, pushing the ES cells in the injection needle into the embryos, and finally pulling out the needle;

(4) transferring the injected embryo to embryo culture medium, culturing at 37 deg.C and 5% CO₂Culturing for 24 hours;

(5) selecting the well-developed embryo in the step (4), and transplanting the well-developed embryo into 2.5d of uterus of a surrogate mouse;

(6) when a pregnant mouse gives birth to a young mouse F0, counting and identifying the born mouse;

the liquid drop in the injection dish in the step (3) is injection, and the modified embryo culture medium in the step (4); they are identical in composition and consist of:

；

wherein the NEAA (100X) comprises the following components:

composition (I) Concentration (g/100mL) Proline 0.01-0.1 Alanine 0.01-0.1 Serine 0.01-0.1 Glycine 0.01-0.1 Asparagine 0.05-0.5 Aspartic acid 0.05-0.5 Glutamic acid 0.05-0.5

Wherein the small molecular substance is amino aromatic hydrocarbons composed of C, H, F, N, O, S chemical components, and the range of the small molecular substance is C (11-25) H (9-30) F (2-8) N (1-10) O (2-8) S (1-5).
2. The method of claim 1, wherein in step (2), the 2-cell embryo is washed in M2 culture drop for multiple times, then the 2-cell embryo is transferred to a micro-droplet culture dish, and then after washing the 2-cell embryo with well-balanced M16 culture solution for multiple times, the 2-cell embryo is finally placed in a clean drop.
3. The method according to claim 1, wherein the step (3) of microinjection comprises in particular the steps of:

s1, selecting a single ES cell under a high power microscope, continuously sucking a plurality of cells into an injection needle by using the injection needle with an inclined opening, selecting an embryo with a better form, blowing and sucking the embryo by using a fixing needle, rotating the embryo until a larger gap is adjusted in the direction of 3 points, fixing the embryo by using the fixing needle under negative pressure, adjusting a microscope to determine the edge of the embryo, and simultaneously ensuring the firm fixation of the embryo;

s2, arranging the tip of the injection needle and the center point of the embryo on the same focusing plane, and lightly contacting the surface of the embryo by using the tip of the injection needle;

s3, inserting a needle head of an injection needle into a blastomere gap of the embryo, slowly pushing an oil pressure injector, pushing the ES cells in the injection needle into the embryo, and injecting 6-8 ES cells into each embryo;

s4, after injection, the injection needle is slowly pulled out.

4. The method according to claim 1, wherein the liquid drop in the injection dish in step (3) is an injection liquid, the modified embryo culture medium in step (4); they are identical in composition and consist of:

name of reagent Adding amount of Sodium chloride 4.0-6.0g/L Potassium chloride 0.1-0.5g/L Calcium chloride 0.1-0.5g/L Potassium dihydrogen phosphate 0.1-0.5g/L Magnesium sulfate 0.1-0.3g/L Sodium bicarbonate 1.0-3.0g/L β -mercaptoethanol 0.1-10mL/L Transferrin 0.005-0.083g/L Glucose 0.1-1.0g/L Sodium lactate 1.0-5.0mL/L Calcium lactate 0.1-1.0g/L Pyruvic acid sodium salt 0.01-0.1g/L Ethylenediaminetetraacetic acid sodium salt 0.01-0.1g/L Glutamine 0.1-0.2g/L Bovine serum albumin 0.001-1g/L Fetal bovine serum 5-10％ HEPES 2.0-5.0g/L NEAA(100×) 0.5-5× Small molecule substances 0.005-10g/L

Wherein the NEAA (100X) comprises the following components:

composition (I) Concentration (g/100mL) Proline 0.05-0.1 Alanine 0.05-0.1 Serine 0.05-0.1 Glycine 0.05-0.1 Asparagine 0.05-0.4 Aspartic acid 0.05-0.4 Glutamic acid 0.05-0.4

Wherein the small molecular substance is amino aromatic hydrocarbons composed of C, H, F, N, O, S chemical components, and the range of the small molecular substance is C (11-25) H (9-30) F (2-8) N (1-10) O (2-8) S (1-5).

5. The method according to claim 1 or 4, wherein the small molecule substance is selected from two or more of the components XAV939, PD184352, A0-6301, PD184352, AZD6244, CT99021, GSK1120212, PS-341.