CN116479052A - Preparation method and application of MTHFR gene modified mice - Google Patents

Abstract

The invention discloses a preparation method of an MTHFR gene modified mouse, wherein the mouse expresses mutant MTHFR protein, and the mutant MTHFR protein comprises an amino acid sequence of any one or more amino acid mutations from position 215 to position 225 of the MTHFR gene.

Description

Preparation method and application of MTHFR gene modified mice

Technical Field

The invention relates to a method for constructing an animal model by gene editing and application thereof, in particular to a method for constructing an MTHFR gene point mutation mouse and application thereof.

Background

Folic acid can be metabolized in vivo through two modes of methylation and sulfur conversion, and genes such as methylene tetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR) and the like which participate in metabolism have polymorphism, so that the concentration of active products of folic acid in a human body is different after the folic acid is supplemented. The gene encoding MTHFR is located at position No.1 chromosome lp36.3, has 11 exons, and has polymorphism. The reductase reduces 5, 10-methylene tetrahydrofolate to 5-methyl tetrahydrofolate in the folate metabolic pathway, which is also the only form of folate in the blood circulation, which is taken up and utilized by cells. Frosst et al found MTHFR 677C > T mutation for the first time in 1995, which resulted in thermal instability, and laid the theoretical foundation of difference in MTHFR different genotypes activity. There are 3 genotypes at MTHFR677 locus, wild type CC, heterozygous mutant CT, homozygous mutant TT, and studies show that about 29% of human MTHFR genes are 677TT in Chinese population.

At present, the MTHFR677 gene polymorphism has been studied more in relation to diseases, including psoriasis, diabetes and diabetes related psychosis, acute coronary syndrome, hypertension, infertility, stroke, mental diseases, cancer, etc. The MTHFR677TT genotype is known to have a negative impact on human health and therefore it is difficult to explain its high proportion of presence in humans. The gene may have specific unique properties and may also have a positive effect on humans. One study showed that MTHFR677TT may provide protection against megaloblastic anemia during periods of inadequate dietary folate supply [ Green R, miller JW.Folate deficiency beyond megaloblastic anemia: hyperhomocysteinemia and other manifestations of dysfunctional folate status.Semin Hematol 1999;36 (1):47-64.]. There are also scholars pointing out that mutations in the relevant genes might be grouped against malaria infection [ Meadows DN, pyzik M, wu Q, et al, incorporated resistance to malaria in mice with methylenetetrahydrofolate reductase (Mthfr) deficiency suggests amechanism for selection of the MTHFR677 c > T (c.665c > T) variant. 35 (5):594-600.].

A good animal model can help people find and study the pathology of 677TT related diseases and find related drugs. In 2001 Chen et al, by using gene targeting technology of embryonic stem cell pathway, it was realized that insertion of a gene of neo-enzymatic phosphotransferase (neo) at the third exon of the mouse MTHFR gene destroyed the activity of MTHFR enzyme [ Chen Z, karaplis AC, ackerman SL, et al Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid position. Hum Mol Genet.2001;10 (5):433-443.]. As a classical MTHFR gene-deficient mouse, it is widely used. However, although the above model produced mice with mild MTHFR deficiency, it was not consistent with the 677TT situation in humans, most of the 677TT population and normal population could live normally, and the related gene mutation did not significantly affect health. There is therefore a need for a tool that is closer to the above situation to study and develop relevant drugs.

Disclosure of Invention

One of the purposes of the invention is to provide a construction method of an MTHFR gene point mutation mouse, wherein the mouse expresses a mutant MTHFR protein, the mutant MTHFR protein comprises a 221 th amino acid mutation of an MTHFR gene shown in SEQ ID NO. 17, the amino acid mutation is Ala mutation to Val, a corresponding base is changed from GCC to GTC, and the method comprises the step of knocking the mutant gene into a target sequence of the MTHFR gene by using a gene editing technology.

The invention uses a gene editing technology to knock in the MTHFR specific site, wherein the gene editing technology is CRISPR/Cas9 technology.

Preferably the construction method comprises inserting the sequence into the MTHFR locus using sgrnas and/or targeting vectors.

Preferably, the 3' -end of the sgRNA comprises a PAM sequence, and more preferably, the PAM sequence may be GGG, TGG, AGG.

Further preferred, the bottom strand oligonucleotide of the sgRNA has more than 80% homology with any of SEQ ID nos. 1 to 16 or comprises the nucleotide sequence shown in any of SEQ ID nos. 1 to 16.

The construction method also comprises a method for obtaining mice with stable genotypes by mating the primary gene editing mice with wild types.

Preferably, the construction method comprises the steps of:

1) Preparing and obtaining a forward oligonucleotide sequence and a reverse oligonucleotide sequence by using any one or more sgRNA target sequences shown as SEQ ID NO. 1-16;

2) Ligating a primer corresponding to the sgRNA sequence into a pCS plasmid vector in a Gibson Assembly mode to construct a Cas9/sgRNA plasmid;

3) Connecting the Cas9/sgRNA expression vector to a plasmid vector with a T7 promoter for in vitro transcription to obtain RNA for injection;

4) Injecting the in vitro transcription product into a mouse cell, wherein the cell is a fertilized egg cell or an embryonic stem cell;

5) And (3) transplanting the cells obtained in the step (4) into oviducts of female mice to develop after culturing, and then identifying the genetic modification condition of the mice after development, screening and transferring the germ line.

It is another object of the present invention to provide an application of a mouse model resembling human MTHFR677TT, the application comprising: 1. application in product development involving the MTHFR gene; 2. use as a model system for toxicology, pharmacology, immunology and medical research; 3. relates to the application of screening, drug effect detection and evaluation of folic acid metabolism drugs; 4. the use of MTHFR gene or protein function was investigated.

It is a third object of the present invention to provide an sgRNA molecule targeting the MTHFR gene.

Preferably, the sgRNA molecule sequence is shown in SEQ ID NO. 1-16.

Further preferably, the sgRNA molecule sequence is shown as SEQ ID NO. 3 or SEQ ID NO. 13.

It is a fourth object of the present invention to provide an MTHFR gene-modified cell or cell line, at least one chromosome of which comprises an MTHFR gene encoding a mutated MTHFR protein, said cell being derived from a mouse cell genetically edited using the sgRNA molecule of the present invention or the point mutation of the present invention.

The cells or cell lines of the invention are not animal varieties and do not develop into individuals.

It is a fifth object of the present invention to provide a drug screening or evaluation method comprising detecting and evaluating effects of folic acid metabolism after administration of a drug using MTHFR modified cells.

The genetic mutation comprises a base substitution, a frame shift, a deletion or an insertion. Preferably, the gene mutation is a base substitution.

The term "homology" as used herein refers to nucleotide sequences, and those skilled in the art can adjust the sequences according to actual working requirements so that the sequences used have 80%,85%, 90%, or more than 95% homology to the sequences described herein.

The mice of the invention are rodents selected from the group consisting of BALB/C, A/He, A/J, A/WySN, AKR, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C BL/A, C BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6J, C BL/6ByJ, C57BL/6NJ, C57BL/10ScSn, C57BL/10Cr and C57BL/Ola of the C57BL, C58, CBA/Br, CBA/Ca, CBA/J, CBA/st, CBA/H strains.

Compared with the common MTHFR-/-mice, the MTHFR gene point mutation mice have the protein function close to that of human MTHFR677TT, so that the physiological functions of related high-risk groups occupying nearly 20% of Chinese population can be estimated and researched more effectively, and the development of related medicaments and treatment methods can be facilitated.

The following examples further illustrate the invention in detail and are not to be construed as limiting the scope of the invention or the particular methods described herein.

Drawings

FIG. 1 schematic representation of MTHFR gene targeting.

FIG. 2 Activity detection results for Cas 9/sgRNA.

FIG. 3EGE-GJ-118-A-sgRNA3 and EGE-GJ-118-A-sgRNA13 were ligated into a T7 promoter-harboring plasmid vector and transcribed in vitro to give an RNA electrophoretogram.

FIG. 4 is a map of a targeting vector.

FIG. 5F 0 mice birth after microinjection of Cas9/sgRNA and targeting vector to mouse fertilized eggs.

FIG. 6 verifies the PCR conditions for the mouse gene case.

FIG. 7 partial identification results of rat tail genotype identification in the F0 generation.

FIG. 8 partial identification results of rat tail genotype identification in the F0 generation.

FIG. 9F1 generation mice genotyping.

FIG. 10F1 shows the result of Southern blot detection of homologous recombination mice.

Detailed Description

It will be understood by those skilled in the art that various modifications and substitutions can be made in the details and form of the present invention without departing from the spirit and scope of the invention.

Example 1: construction of MTHFR Gene mutant mice

The mouse MTHFR Gene (NCBI Gene ID:17769, located on the positive strand of chromosome 4, approximately 21.2kb in length). For the purposes of the present invention, 1 or more point mutations can be introduced into the coding sequence at the mouse MTHFR locus, resulting in an MTHFR mutant gene that encodes a protein sequence having 1 or more amino acid mutations compared to the wild type protein.

Specifically, according to the characteristics of the MTHFR gene, the function of the mutation of the humanized MTHFR677 gene is analyzed, and the situation that the 221 th amino acid of the mouse is closest to the 221 th amino acid of the human MTHFR677TT after mutation, namely, the 221 th amino acid Ala of the mouse is mutated into Val, and the corresponding base is changed from GCC to GTC is determined.

The CRISPR/Cas system is introduced for gene editing, the schematic diagram is shown in figure 1, and the target sequence in the system determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cut a target gene, so that the high-efficiency specific targeting sequence selection and design are the precondition for constructing the sgRNA expression vector.

In order to ensure the consistency of the target gene sequence with the disclosure to ensure the efficiency of the designed Cas 9/sgrnas, PCR amplification and sequencing verification of the C57BL/6N mouse tail target site sequence is first required to ensure the complete consistency of the sgRNA recognition sequence with the C57BL/6N mouse tail DNA sequence. PCR and sequencing are carried out on C57BL/6N rat tail DNA, and the results prove that: the C57BL/6N rat tail target sequence was identical to the sequences given by Genebank and Ensembl.

The sgRNA sequence that recognizes the target site is designed and synthesized. The target site is positioned on amino acid 221 of the MTHFR gene, 16 sgRNAs are designed in the target site region based on the design principle of the sgRNAs, and the corresponding target sequences are as follows:

according to 5' design

MTHFR-sgRNA target site sequence (SEQ ID NO: 1): 5'-AGCTTTGTGGCAGCCTCGGTGGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 2): 5'-TTGCAGTCTAATTACACAAGGGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 3): 5'-GAAAGGATGGGAATCACGTTGGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 4): 5'-GGTGCCAGTCTCTGTAGATTTGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 5): 5'-GTGATTCCCATCCTTTCAGATGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 6): 5'-ACTCATTAGAAGCCTTAAACAGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 7): 5'-AGTCTAATTACACAAGGGGATGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 8): 8'-TAATTACACAAGGGGATGGGCGG-3'

According to 3' design

MTHFR-sgRNA target site sequence (SEQ ID NO: 9): 5'-CAGTATCATTAAGGCGGAATAGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 10): 5'-TTAATCGGAGTCTCTTCACTTGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 11): 5'-TCATCAGATTGCCTTAGATTCTGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 12): 5'-ATCATTAAGGCGGAATAGGCAGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 13): 5'-TCCATGGAGAATTTCAGTATGGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 14): 5'-TTCCATGGAGAATTTCAGTATGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 15): 5'-CAGAGTCCACTCCAGAATCTAGG-3'

MTHFR-sgRNA target site sequence (SEQ ID NO: 16): 5'-CCTTTGACAATGCAATTAATCGG-3'

Corresponding primers were synthesized according to the designed sgRNA sequence, ligated into pCS-4G vector by means of Gibson Assembly, and the ligation products were transformed and confirmed to be correct by sample sequencing.

The UCA kit is used for detecting the activities of a plurality of sgRNAs, the result shows that the sgRNAs have different activities, according to our experiments and specific activity values, the result is shown in figure 2, and finally, the MTHFR-sgRNA target site sequence (SEQ ID NO: 3) and the MTHFR-sgRNA target site sequence (SEQ ID NO: 13) are selected for the next experiment, and the constructed expression vectors are named as EGE-GJ-118-A-sgRNA3 and EGE-GJ-118-A-sgRNA13 respectively. EGE-GJ-118-A-sgRNA3 and EGE-GJ-118-A-sgRNA13 were ligated into a T7 promoter-harboring plasmid vector and subjected to in vitro transcription (transcription was performed according to the instruction method using the Ambion in vitro transcription kit) to obtain microinjected RNA. The RNA electrophoresis pattern is shown in FIG. 3.

The targeting vector construction can be carried out by conventional methods, such as enzyme digestion, ligation, direct synthesis and the like. After the constructed targeting vector is subjected to primary verification through enzyme digestion, the targeting vector is sent to a sequencing company for sequencing verification, and the map of the targeting vector is shown in figure 4. The targeting vector sequence is shown in SEQ ID NO. 18, and the targeting vector plasmid with the correct sequencing verification is used for subsequent experiments.

Taking a prokaryotic fertilized egg of a mouse, for example, a fertilized egg of a C57BL/6N mouse, premixing Cas9/sgRNA and a targeting vector by using a microinjection instrument, and injecting the premixed fertilized egg into cytoplasm or nucleus of the fertilized egg of the mouse. Microinjection of fertilized eggs was performed according to the method of the "mouse embryo handling laboratory Manual (third edition)" (Andella Naji, chemical industry Press, 2006), and fertilized eggs after injection were transferred to a culture medium for brief culture and then transplanted to oviducts of recipient mice for development. The F0 mice were born after injection as shown in figure 5.

The obtained mice (F0 generation) are crossed and selfed to expand population quantity, and stable MTHFR gene mutant mouse strain is established. The Cas9/sgRNA injection fertilized egg method constructs a knock-in mouse. Since embryo early cleavage is fast, the resulting F0 mice are chimeras. Therefore, the F0 genotype obtained by the identification of the tail of the F0 mouse is only used as a reference, and cannot represent that the F0 genotype is necessarily a heritable gene mutant type, and the heritable genotype is required to be determined after the tail of the F1 mouse is detected.

The genotype of the somatic cells of the F0 mice can be identified by conventional detection methods, e.g., the F0 mouse tail can be identified by PCR methods, and the correct PCR bands can be identified for recovery sequencing to confirm whether accurate recombination of the point mutations has occurred. The specific PCR primers designed were as follows:

EGE-GJ-118-L-GT-F:5’-AACCGGCTAGGAAGCACTGCTAAAG-3’(SEQ ID NO:19)

EGE-GJ-118-L-GT-R:5’-TCTCTAAAGCCCATATGATCAAGTACTC-3’(SEQ ID NO:20)EGE-GJ-118-R-GT-F:5’-CTCTCTAGCCCCAACGATATCAATATTG-3’(SEQ ID NO:21)EGE-GJ-118-R-GT-F:5’-TCACCAGCCAATCATCTGCCAAACT-3’(SEQ ID NO:22)

the PCR conditions are shown in FIG. 6.

The partial identification results are shown in fig. 7 and 8.

Conclusion: EJ118-040, EJ118-044 and EJ118-054 were positive F0 mice as indicated by PCR products and sequences. And (5) selecting positive mice in the rat tail genotype identification result of the F0 generation to mate with the wild type to obtain F1 generation mice with stable genotypes. F1 mice were identified according to the F0 genotype identification method, and the results are shown in FIG. 9. The results obtained by Southern blot detection are shown in FIG. 10, and indicate that 1EJ118-007, 1EJ118-008, 1EJ118-009 and 1EJ118-022 are F1 positive mice. This shows that the method of the invention can be used to construct genetically engineered mice which can be stably passaged and have accurate point mutation of target genes, all mice and offspring have white whole body hair, and compared with normal mice, no obvious abnormality is observed in vitro.

Example 2: application of MTHFR gene engineering mouse

Taking the mouse embryo stem cell prepared by the invention, carrying out DNA recombination identification by using a Southern Blot technology, and confirming that the MTHFR gene in the cell has the predicted mutation. Under the environment of different drugs, the LC-MS/MS technology is used to detect the metabolites of different folic acids in cells, and determine the folic acid metabolism effect of different drugs on MTHFR gene defective cells, so as to evaluate the effect of the drugs on human MTHFR677TT population.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims (12)

1. A preparation method of an MTHFR gene modified mouse is characterized in that the mouse expresses mutant MTHFR protein, and the mutant MTHFR protein comprises protein expressed by an amino acid sequence of any one or more amino acid mutations at positions 215-225 of the MTHFR gene shown in SEQ ID NO. 17.

2. The method according to claim 1, wherein the mutated MTHFR protein is a protein expressed by an amino acid sequence mutated from amino acid 221 of the MTHFR gene, and further wherein the amino acid is mutated from Ala to Val, and the corresponding base is changed from GCC to GTC.

3. The method according to any one of claims 1 to 2, wherein the MTHFR gene modified mice are prepared by introducing 1 or more mutations at the endogenous MTHFR locus of the mice.

4. A method of preparation according to any one of claims 1-3, characterized in that the method comprises knocking the mutant gene into the target sequence of the MTHFR gene using gene editing techniques, preferably the gene editing technique is CRISPR/Cas9 technique, preferably the method of preparation comprises inserting the sequence into the MTHFR locus using sgRNA and/or targeting vectors.

5. The preparation method of the MTHFR gene modified mouse according to any one of claims 1 to 4, wherein the preparation method comprises the steps of introducing one or more point mutations into the amino acid sequences at positions 215 to 225 of the mouse MTHFR gene locus by using sgRNA and/or a targeting vector; the sgRNA targeting target site sequence is shown as SEQ ID NO: 1-16.

6. A method for preparing an MTHFR gene modified mouse, comprising the steps of:

1) Preparing and obtaining a forward oligonucleotide sequence and a reverse oligonucleotide sequence by using any one or more sgRNA target sequences shown as SEQ ID NO. 1-16;

2) Ligating a primer corresponding to the sgRNA sequence into a pCS plasmid vector in a Gibson Assembly mode to construct a Cas9/sgRNA plasmid;

3) Connecting the Cas9/sgRNA expression vector to a plasmid vector with a T7 promoter for in vitro transcription to obtain RNA for injection;

4) Injecting the in vitro transcription product into a mouse cell, wherein the cell is a fertilized egg cell or an embryonic stem cell;

5) And (3) transplanting the cells obtained in the step (4) into oviducts of female mice to develop after culturing, and then identifying the genetic modification condition of the mice after development, screening and transferring the germ line.

7. The sgRNA targeting MTHFR gene is characterized in that the sequence of a target site targeted by the sgRNA is shown as SEQ ID NO: 1-16.

8. A mutant MTHFR protein, wherein the mutant MTHFR protein comprises an amino acid sequence having any one or more of amino acid mutations at positions 215-225, preferably wherein the mutant MTHFR protein comprises an amino acid mutation at position 221 of the MTHFR gene, wherein the amino acid mutation is an Ala mutation to Val mutation. .

9. An MTHFR gene encoding the mutant mouse MTHFR protein of any of claims 1 to 6, wherein the MTHFR gene sequence is a mouse amino acid 221 mutation, preferably the corresponding base is changed from GCC to GTC.

10. A mouse cell or tissue, wherein the cell or tissue expresses a mutated MTHFR protein selected from the group consisting of the mutated MTHFR proteins of claim 8.

11. The method according to any one of claims 1 to 6, wherein the mouse is selected from the group consisting of BALB/C, A/He, A/J, A/WySN, AKR, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6J, C BL/6ByJ, C57BL/6NJ, C57BL/10ScSn, C57BL/10Cr and C57BL/Ola, and the group consisting of C57BL, C58, CBA/Br, CBA/Ca, CBA/J, CBA/st and CBA/H.

12. Use of MTHFR gene modified mice derived from the preparation method of any one of claims 1 to 6, mutant mouse MTHFR proteins of claim 8, mouse MTHFR genes of claim 9, mouse cells and tissues of claim 10 in the development of products involving MTHFR genes, as model systems for toxicology, pharmacology, immunology and medical research, screening, pharmacodynamics detection, evaluation of folic acid metabolism drugs, use of MTHFR genes or protein functions.