CN115029382A - Preparation method of genetically modified mouse capable of distinguishing Tbx1 two variable spliceosomes - Google Patents
Preparation method of genetically modified mouse capable of distinguishing Tbx1 two variable spliceosomes Download PDFInfo
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Abstract
The invention provides a targeting vector capable of distinguishing Tbx1 two variable spliceosomes and a preparation method of a genetically modified mouse thereof, which comprises the steps of designing and constructing the targeting vectors of Tbx 1201 and 203 variable spliceosomes, selecting a first intron of Tbx 1201 and 203 as editing sites, and injecting the targeting vector, guide RNA and Cas9 protein into a mouse fertilized egg together by microinjection. The genetically modified mouse can accurately distinguish two splicing bodies of Tbx1, and the heart outflow tract development of the Tbx 1203 mutation homozygous mouse has obvious defects, so that a reliable animal model is provided for disclosing the action mechanism of the two variable splicing bodies of Tbx1 in the process of congenital heart disease.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a targeting vector capable of distinguishing two variable spliceosomes of Tbx1 and a preparation method of a genetically modified mouse of the targeting vector.
Background
Defects in early cardiac development are the leading cause of early embryonic death, accounting for 30% of cases of human embryonic and early fetal death. The research of the pathogenesis model of the congenital heart disease provides guarantee for solving more and more complex biological problems, and simultaneously avoids partial problems of ethical morality and technical limitation. Mice are very suitable as animal models of the pathogenesis of congenital heart disease, because the development process of the heart of mice is highly similar to the development process of human embryos, and the mice have four-chamber structures as well as human embryos, and have the advantages of strong reproductive capacity, short reproductive cycle, stable genetic background and mature operating system, so the mice become ideal models for genetic research of congenital heart disease.
Tbx1 is one of the members of the T-box family of transcription factors. Studies have reported that Tbx1 is a candidate gene for 22q11.2 microdeletion syndrome (22 q11 deletion syndrome, 22q 11.2ds) using a Tbx1 gene knockout mouse model. Specifically, mild defects in the cardiac outflow tract (OFT) were observed in Tbx1 +/-mice, Tbx 1-/-mice showed more severe defects in the heart, craniofacial, thymus and parathyroid glands, and the associated phenotypes were consistent with DiGeorge syndrome (DGS)/palatal facial syndrome (VCFS) patients. In addition, mouse gene dose studies show that Tbx1 belongs to a dose-dependent gene, the expression of the gene is different in space and time in different tissues, and the expression of Tbx1 needs to be accurately regulated and controlled during embryonic heart development. Notably, the reported Tbx1 knockout mice are common domain knockouts for variable spliceosomes and do not accurately distinguish between the roles and mechanisms of different variable spliceosomes during development. In other words, the Tbx1 knockout mice reported at present can prove the main function and the regulatory network of the gene, but cannot distinguish the action and mechanism of different variable splicing bodies of Tbx1 in the heart development process. Therefore, there is a need for a mouse model that can accurately differentiate between studies of different splice bodies of Tbx1, and provide an effective tool for revealing the different splice bodies in terms of pathogenic mechanisms.
Compared with Zinc Finger Nucleases (ZFNs) and transcription activator-like effector nucleases (TALEN) technologies, the CRISPR/Cas9 gene editing technology has the advantages of short cycle, high efficiency and strong specificity. Meanwhile, expression forms of different variable shears of Tbx1 are accurately distinguished by means of different tag proteins of V5 and 3xFlag, deletion of corresponding shears is realized through point mutation, conditional knockout of Tbx 1201 and Tbx 1203 can be realized through a Cre-loxp system, and acquisition of related mice has important significance for disclosing action mechanisms of different variable shears of Tbx1 in the heart development process.
Disclosure of Invention
The invention aims to provide a targeting vector capable of distinguishing two variable splicing bodies of Tbx1 and a preparation method of a genetically modified mouse thereof.
The invention provides a preparation method of a genetically modified mouse capable of distinguishing two variable spliceosomes of Tbx1, which is characterized by comprising the following steps:
designing and constructing a targeting vector of Tbx 1201 and 203 variable cleavers;
selecting the Tbx 1201 and the intron 203I as editing sites;
the targeting vector, guide RNA and Cas9 protein were co-injected into mouse zygotes by microinjection.
Further, the targeting vector for distinguishing two variable splicing bodies of Tbx1 and the preparation method of the mouse modified by the gene of the targeting vector also have the following characteristics: the gene modified mice were screened for progeny positive mice by PCR and gene sequencing methods.
Further, the targeting vector for distinguishing two variable splicing bodies of Tbx1 and the preparation method of the mouse modified by the gene of the targeting vector also have the following characteristics: the guide RNA sequence 1 matched to Tbx1 variable cutter is: CTTTCGGTCAAGGACTTAGTAGG; guide RNA sequence 2 matched to Tbx1 variable cutter was: CTGGGACTCTTCGAATTCGAGGG
Further, the targeting vector for distinguishing two variable splicing bodies of Tbx1 and the preparation method of the mouse modified by the gene of the targeting vector also have the following characteristics:
embryo isolation and microinjection procedure:
1) on the first day, selecting 3-4 weeks old C57BL/6J female mice, injecting 5 IU pregnant horse serum gonadotropin (PMSG) into the abdominal cavity of each mouse, injecting 5 IU Human Chorionic Gonadotropin (HCG) into the abdominal cavity after 48 hours, and mating with male mice of 2-8 months immediately after injection;
2) the following day, 9 am, before examination of the mouse pessaries, donor females and males were separated;
3) on the fourth day, fertilized eggs were collected from the oviducts of female mice, and cumulus cells were placed in M2 medium containing 0.1% bovine hyaluronidase. Releasing fertilized eggs, and culturing in a 5% CO2 incubator at 37 ℃;
4) selecting fertilized eggs with normal shapes and clear pellucida and male pronuclei for microinjection under an inverted microscope;
5) sucking the fertilized egg by using a holding capillary, and injecting a targeting vector, guide RNA and Cas9 protein which are identified correctly by enzyme digestion into the cytoplasm of the fertilized egg together;
6) microinjecting 300-400 fertilized eggs, and culturing the fertilized eggs after injection in an M16 (Sigma) culture medium in a 5% CO2 incubator at 37 ℃ for 1 hour;
7) preparing a 3-6 month-old female mouse for surrogate pregnancy one day before microinjection, injecting 120 mg/kg ketamine and 16 mg/kg xylazine into the abdominal cavity to anaesthetize the female mouse for surrogate pregnancy, and placing the female mouse on a constant temperature table;
8) shearing the skin of the abdominal cavity of the mouse by using sterile scissors, pulling out the uterine horn of the pregnant female mouse, maintaining the humidity by using 0.9 percent NaCl solution, and fixing the uterus;
9) forceps are used to clamp the upper end of ampulla of fallopian tube, and a small hole is formed on the wall of fallopian tube. Injecting 20-30 fertilized eggs into the oviduct of each surrogate female mouse by using a thin glass capillary;
10) putting the uterus into the mouse body again, and putting the mouse into a breeding cage for breeding after surgical suture;
11) after the mouse is born for 2-3 weeks, shearing ear numbers, shearing tails, extracting a genome, amplifying fragments by PCR, and carrying out gene sequencing identification to obtain a positive F0-generation mouse;
12) sexually mature positive F0 mice were mated with wild type C57BL/6J mice to obtain F1 mice, and progeny mice of about 2 weeks after birth were tailed to extract genomes and identified by PCR using primer sequence 3 and primer sequence 4.
Further, the targeting vector for distinguishing two variable cleavants of Tbx1 and the method for preparing the genetically modified mouse thereof according to the present invention are characterized by further comprising:
the identification process of mouse genotype:
1) shearing 0.1-0.2 cm of mouse tail into 1.5 ml of EP tube, adding 150 μ l NaOH (50mM) lysis buffer;
2) ensuring that the lysate submerges the sample, and incubating overnight in a constant-temperature water bath kettle at 65 ℃;
3) centrifuging at 12000 rpm for 2 min, and sucking the supernatant;
4) adding 20. mu.l of Tris-HCl (1M, pH7.5), and fully mixing;
5) phenol: chloroform: adding 150 mul of mixed solution into isoamyl alcohol =25:24:1, and uniformly mixing by vortex oscillation;
6) centrifuging at 12000 rpm for 2 min, and sucking the supernatant into a new EP tube;
7) adding 1 ml of precooled absolute ethyl alcohol, fully and uniformly mixing, centrifuging at 4 ℃ and 12000 rpm for 10 minutes, and removing supernatant;
8) drying in an ultraclean bench, dissolving the precipitate with 100. mu.l of double distilled water, and taking 2. mu.l as a template for PCR identification.
Further, the targeting vector for distinguishing two variable cleavants of Tbx1 and the method for preparing the genetically modified mouse thereof according to the present invention are characterized by further comprising:
the positive mice were designated TMV (Tbx 1201 flox-V5-G15) and TMF (Tbx 1203 flox-3 xFlag-C9), respectively, as judged by F0 mouse PCR products:
1) the TMV positive mouse contains 3.0 kb and 4.1 kb fragments after being amplified by TMV-1 in the primer sequence 1, and the product size after TMV-2 amplification is 6.2 kb. Only 4.1 kb fragment is obtained after TMV-1 amplification, and no fragment obtained after TMV-2 amplification is a negative mouse.
2) TMF positive mice amplified a 4.9 kb fragment by TMF-1 in primer sequence 2, TMF-2 amplified a 4.8 kb fragment, and negative mice amplified no fragment.
The primer sequences for PCR amplification of F0 mouse were as follows:
TMV-1F：5’ -ACAGGCGGTGCTTGTCTTAG-3’
TMV-1R：5’ -AAGAGAGGCGATGCTGAACG-3’
TMV-2F：5’ -AGCATCGCCTCTCTTAAGTCC-3’
TMV-2R：5’ -GCCTCTGATGGGGAGTTTCC-3’
TMF-1F：5’ -GCAAGATTTGCAGCTTATTAGCC-3’
TMF-1R：5’ -GTGGATTCGGACCAGTCTGA-3’
TMF-2F：5’ -ACGTAAACGGCCACAAGTTC-3’
TMF-2R：5’ -TGGATTTGGCGTCACTAGCCAG-3’
Further, the targeting vector for distinguishing two variable cleavants of Tbx1 and the preparation method of the genetically modified mouse thereof are characterized by further comprising the following steps:
determination of the sizes of PCR products of offspring mice:
1) in the PCR product of the TMV mouse genome primer sequence 3, only 223 bp of the fragment size is homozygote mouse, 181 bp of the fragment size is heterozygote mouse, and the rest is wild mouse.
2) In the PCR product of the TMF mouse genome primer sequence 4, the fragment with the size of only 360 bp is a homozygote mouse, meanwhile, the fragment with 298 bp is a heterozygote mouse, and the rest is a wild type mouse.
The sequences of primers used for PCR amplification of progeny mice are as follows:
primer sequence 3:
TMV-3F：5’ -CGTTCAGCATCGCCTCTCTT-3’
TMV-3R：5’ -GCCTGACAGTATAGACGCGG-3’
primer sequence 4:
TMF-3F：5’ -TGAAAAGCGGATGAAGGTGCAG-3’
TMF-3R：5’ -GGAAAATGAGCGCAATGGCTTTTA-3’
drawings
Figure 1 shows the location of the Tbx 1201 and Tbx 1203 splice body loci on the genome.
FIGS. 2A and 2B are schematic diagrams of the construction strategies associated with TMV and TMF genetically modified mice.
FIGS. 3A and 3B are the maps of the TMV and TMF gene modified mouse homologous recombination donor vectors.
FIGS. 4A and 4B show the results of enzyme digestion identification of the homologous recombinant donor vector of TMV and TMF genetically modified mice.
FIGS. 5A and 5B are the PCR identification electrophoretograms of 5 'homology arm and 3' homology arm of mice genetically modified with TMV and TMF at the F0 generation.
Fig. 6A and 6B are the identification of TMV and TMF mouse PCR products at generation F1.
Fig. 7A and 7B are the identification of TMV and TMF mouse PCR products at generation F2.
FIG. 8 is a front view of the heart of a TMF, TMV homozygous mouse generation F2.
Detailed Description
The technical solution of the present invention will be further described with reference to the following embodiments.
The invention selects a Tbx1 gene sequence in a mouse chromosome 16 to design a homologous arm to construct a targeting vector, adds V5 and 3xFlag tag proteins near the initiation codon ATG of two variable splicing bodies of Tbx 1201 and 203, and introduces point mutation in an exon I of the variable splicing bodies to respectively knock out the Tbx 1201 and 203. By adding flox to both sides of the exon containing Tbx 1201 and 203, a co-knock-out of Tbx 1201 and 203 can be achieved by breeding with mice expressing Cre. Obtaining an editing mouse by using a CRISPR/Cas9 technology, respectively selecting a first intron of a variable splice as an editing site, transcribing guide RNA in vitro, co-injecting a targeting vector, the guide RNA and a Cas9 protein into a mouse fertilized egg by using microinjection, and screening a positive mouse from offspring by using a PCR (polymerase chain reaction) and gene sequencing method. The generated genetically modified mouse carries V5 and 3xFlag tag proteins and can be recognized by corresponding antibodies, so that an effective strategy and a scheme are provided for solving the problem that the current Tbx1 antibody cannot distinguish the type of the Tbx1 variable spliceosome.
One, gene modified site and guide RNA
1. Site of gene modification
1.1 the mouse Tbx1 gene (GenBank accession No.: NM-001285472.1, Ensembl database No.: 00000009097) is located on mouse chromosome 16.
1.2 Tbx1 Gene site has 5 variable cleavages.
1.3 Tbx 1201 variable splice variant (ENSMUST 00000009241.7) contains 9 exons in total, and Tbx 1203 variable splice variant (ENSMUST 00000232335.2) contains 7 exons in total.
1.4 in the modification, flox sequences are added on two sides of the first exon of Tbx 1201 and 203 variable spliceosome, V5 tag protein is added near the ATG of the initiation codon of the Tbx 1201 variable spliceosome, and G base is removed at the 15 th position after the ATG of the initiation codon of the Tbx 1203 variable spliceosome, so that point mutation is caused.
1.5 in the modification, flox sequences are added on two sides of the first exon of the Tbx 1201 variable splice body and the 203 variable splice body, 3xFlag tag protein is added after the initiation codon ATG of the Tbx 1203 variable splice body, and C base is removed at the 9 th position after the initiation codon ATG of the Tbx 1201 variable splice body to cause point mutation.
1.6 this modification will design a targeting vector, the homology arms obtained by PCR amplification using the BAC clone, RP24-78F18 as template.
1.7 the modified TMV targeting vector is identified by SacI restriction enzyme digestion, and the TMF targeting vector is identified by Asc1, Mef 1 and Nhe1 restriction enzyme digestion.
1.8 this modification will construct 2 pairs of guide RNA and ensure correct sequence by sequencing.
1.9 this modification selects the Tbx 1201 and Tbx 1203 variable splice I intron numbers as editing sites.
1.10Cas9 protein and in vitro transcribed guide RNA were co-injected with targeting vector into mouse zygotes.
1.11 the offspring mice born will be screened positive mice by analyzing the genotype through PCR and gene sequencing method.
2. Genetic modification
2.1 schematic diagram
The positions of the different variable cleavants of Tbx1 on the genome are shown in FIG. 1, exons are arranged from left to right, the total length of the Tbx 1201 variable cleavant is about 8.9 kb, the total length of the Tbx 1203 variable cleavant is about 5.3 kb, and the open reading frame ORFs are indicated by solid bars in FIG. 1.
2.2Guide RNA sequences
The guide RNA sequence 1 matched to Tbx1 variable cutter is: CTTTCGGTCAAGGAC TTAGTAGG
Second, embryo separation and microinjection process
1) On the first day, selecting 3-4 weeks old C57BL/6J female mice, injecting 5 IU pregnant horse serum gonadotropin (PMSG) into the abdominal cavity of each mouse, injecting 5 IU Human Chorionic Gonadotropin (HCG) into the abdominal cavity after 48 hours, and mating with male mice of 2-8 months immediately after injection;
2) the following day, 9 am, before examination of the mouse pessaries, donor females and males were separated;
3) on the fourth day, fertilized eggs were collected from the oviducts of female mice, and cumulus cells were placed in M2 medium containing 0.1% bovine hyaluronidase. Releasing fertilized eggs, and culturing in a 5% CO2 incubator at 37 ℃;
4) selecting fertilized eggs with normal shapes and clear pellucida and male pronuclei for microinjection under an inverted microscope;
5) sucking the fertilized egg by using a holding capillary, and injecting the targeting vector, guide RNA and Cas9 protein which are correctly identified by enzyme digestion into the cytoplasm of the fertilized egg together;
6) microinjecting 300-400 fertilized eggs, and culturing the fertilized eggs after injection in an M16 (Sigma) culture medium in a 5% CO2 incubator at 37 ℃ for 1 hour;
7) preparing a 3-6 month-old female mouse for surrogate pregnancy one day before microinjection, injecting 120 mg/kg ketamine and 16 mg/kg xylazine into the abdominal cavity to anaesthetize the female mouse for surrogate pregnancy, and placing the female mouse on a constant temperature table;
8) shearing the skin of the abdominal cavity of the mouse by using sterile scissors, pulling out the uterine horn of the pregnant female mouse, maintaining the humidity by using 0.9 percent NaCl solution, and fixing the uterus;
9) the upper end of the ampulla of the fallopian tube is clamped by a pair of tweezers, and a small hole is formed on the wall of the fallopian tube. Injecting 20-30 fertilized eggs into the oviduct of each surrogate female mouse by using a thin glass capillary;
10) putting the uterus into the mouse body again, and putting the mouse into a breeding cage for breeding after surgical suture;
11) after the mouse is born for 2-3 weeks, shearing ear numbers, shearing tails, extracting a genome, amplifying fragments by PCR, and carrying out gene sequencing identification to obtain a positive F0-generation mouse;
12) sexually mature positive F0 mice are respectively mated with wild C57BL/6J mice to obtain F1 mice, progeny mice about 2 weeks after birth are subjected to tail shearing to extract genomes, and PCR identification is carried out by using a primer sequence 3 and a primer sequence 4.
Third, mouse genotype identification
1. Sample treatment:
1) 0.1-0.2 cm of the mouse tail was sheared into 1.5 ml EP tubes and 150. mu.l NaOH (50mM) lysis buffer was added.
2) The lysate is kept over the sample and incubated overnight in a thermostat water bath at 65 ℃.
3) Centrifuge at 12000 rpm for 2 minutes and aspirate the supernatant.
4) Add 20 μ l Tris-HCl (1M, pH7.5) and mix well.
5) Phenol: chloroform: isoamyl alcohol =25:24:1, 150 μ l of the mixture was added, vortexed, shaken and mixed well.
6) Centrifuge at 12000 rpm for 2 minutes and aspirate the supernatant into a new EP tube.
7) 1 ml of precooled absolute ethanol is added, fully and uniformly mixed, centrifuged at 12000 rpm for 10 minutes at 4 ℃ and the supernatant is discarded.
8) Drying in an ultraclean bench, dissolving the precipitate with 100. mu.l of double distilled water, and taking 2. mu.l as a template for PCR identification.
Enzyme digestion identification of Donor vector
As can be seen from the results of the enzyme digestion identification of the TMV Donor vector SacI in FIG. 4A, the sizes of the fragments after enzyme digestion are respectively 8.5 kb, 4.0 kb, 1.4 kb and 0.8 kb, which is in line with the expectation, and the successful construction of the TMV Donor vector is proved. A10.5 kb fragment is obtained after the TMF Donor vector in FIG. 4B is subjected to enzyme digestion by Asc1, the sizes of the fragments after enzyme digestion by Mfe1 and Nhe1 are 6.3 kb, 2.2 kb, 1.4 kb and 0.7 kb, and the construction of the TMF Donor vector is proved to be successful.
CRISPR-induced modification detection
And carrying out PCR amplification on target regions of different variable splice bodies of the mouse Tbx1 through specific primers, and carrying out sequencing verification on an amplification product to ensure that the editing position of the genome is correct.
The primer sequences for PCR amplification of F0 mouse were as follows:
TMV-1F：5’ -ACAGGCGGTGCTTGTCTTAG-3’
TMV-1R：5’ -AAGAGAGGCGATGCTGAACG-3’
TMV-2F：5’ -AGCATCGCCTCTCTTAAGTCC-3’
TMV-2R：5’ -GCCTCTGATGGGGAGTTTCC-3’
TMF-1F：5’ -GCAAGATTTGCAGCTTATTAGCC-3’
TMF-1R：5’ -GTGGATTCGGACCAGTCTGA-3’
TMF-2F：5’ -ACGTAAACGGCCACAAGTTC-3’
TMF-2R：5’ -TGGATTTGGCGTCACTAGCCAG-3’
The sequences of primers used for PCR amplification of progeny mice are as follows:
primer sequence 3:
TMV-3F：5’ -CGTTCAGCATCGCCTCTCTT-3’
TMV-3R：5’ -GCCTGACAGTATAGACGCGG-3’
primer sequence 4:
TMF-3F：5’ -TGAAAAGCGGATGAAGGTGCAG-3’
TMF-3R：5’ -GGAAAATGAGCGCAATGGCTTTTA-3’
size of PCR amplification product:
TMV wild type gene use: the primer sequence 3, the product length is 181 bp.
TMV heterozygous gene use: the primer sequence 3, the product length is 181 bp and 223 bp.
TMV homozygous gene use: the primer sequence 3, the product length is 223 bp.
TMV wild type gene use: primer sequence 4, and the product length is 298 bp.
TMV heterozygous gene use: primer sequence 4, and the product length is 298 bp and 360 bp.
TMV homozygous gene use: primer sequence 4, and the product length is 360 bp.
The PCR products are shown in FIG. 5A and FIG. 5B.
As shown in FIG. 5A, the gel electrophoresis results of the TMV mouse PCR products revealed that the PCR products of the 5 'arm of the F0 mouse 3, 4, 8, and 9 contained bands of 4.1 kb and 3.0 kb, and the PCR product of the 3' arm contained a band of 6.2 kb, indicating that the above mouse was a heterozygous mouse.
As is clear from the results of gel electrophoresis of the TMF mouse PCR products, the PCR products of the 5 '-arm of mice No. 29, 31 and 39 at the F0 contained a band of 4.9 kb and the PCR product of the 3' -arm contained a band of 6.2 kb, demonstrating that these mice were heterozygous.
As shown in FIG. 6A, the results of the detection of TMV mice at the F1 generation revealed that the PCR products of mice Nos. 3, 4, 8, 9 and 10 contained fragments of 223 bp and 181 bp, which confirmed that these mice were heterozygous mice.
As shown in FIG. 6B, the results of the detection of TMF mice of the F1 generation revealed that the PCR products of mice Nos. 1, 2, 3 and 4 contained fragments of 360 bp and 298 bp, which confirmed that these mice were heterozygous mice.
As shown in FIG. 7A, the detection results of TMV mice of generation F2 show that the PCR products of mice No. 11, 13, 14 and 16 contain 223 bp and 181 bp fragments, which proves that the mice are heterozygote mice; mouse No. 12 only contains 223 bp fragment is homozygous mouse, the rest is wild type mouse.
As shown in FIG. 7B, the result of the detection of TMF mice of the F2 generation revealed that the PCR products of mice No. 6 and 8 contained fragments of 360 bp and 298 bp, which confirmed that these mice were heterozygous mice. Mouse No. 7 contains only a 360 bp fragment as a homozygous mouse, and the remaining mouse No. 5 is a wild-type mouse.
As can be seen from FIG. 8, the outflow tracts of TMF homozygous mice (left) born on the first day (P0) were divided into aorta and pulmonary artery, and no significant abnormality was observed; the aorta of TMV homozygous mice (right panel) born on day one (P0) fused with the pulmonary arteries, and the cardiac outflow tracts appeared markedly abnormal.
Claims (7)
1. A targeting vector capable of distinguishing Tbx1 two variable spliceosomes and a preparation method of a genetically modified mouse thereof are characterized in that:
designing and constructing a targeting vector of Tbx 1201 and 203 variable cleavers;
selecting the Tbx 1201 and the intron 203I as editing sites;
the targeting vector, guide RNA and Cas9 protein were co-injected into mouse zygotes by microinjection.
2. The targeting vector and the method for preparing the genetically modified mouse thereof for differentiating two variable cleavers of Tbx1 according to claim 1, wherein the targeting vector comprises:
the gene modified mice are screened for progeny positive mice by PCR and gene sequencing methods.
3. The targeting vector and the method for preparing the genetically modified mouse thereof for differentiating two variable cleavers of Tbx1 according to claim 3, wherein the targeting vector comprises:
the guide RNA sequence 1 matched to Tbx1 variable cutter is: CTTTCGGTCAAG GACTTAGTAGG
Guide RNA sequence 2 matched to Tbx1 variable cutter was: CTGGGACTCTTC GAATTCGAGGG are provided.
4. The targeting vector and the method for preparing the genetically modified mouse thereof for differentiating two variable cleavers of Tbx1 according to claim 1, wherein the targeting vector comprises:
embryo isolation and microinjection procedure:
1) on the first day, selecting 3-4 weeks old C57BL/6J female mice, injecting 5 IU pregnant horse serum gonadotropin (PMSG) into the abdominal cavity of each mouse, injecting 5 IU Human Chorionic Gonadotropin (HCG) into the abdominal cavity after 48 hours, and mating with male mice of 2-8 months immediately after injection;
2) the following day, 9 am, before examination of the mouse pessaries, donor females and males were separated;
3) on the fourth day, fertilized eggs were collected from the oviducts of female mice, and cumulus cells were placed in M2 medium containing 0.1% bovine hyaluronidase; releasing fertilized eggs, and culturing in a 5% CO2 incubator at 37 ℃;
4) selecting fertilized eggs with normal shapes and clear pellucida and male pronuclei for microinjection under an inverted microscope;
5) sucking the fertilized egg by using a holding capillary, and injecting the targeting vector, guide RNA and Cas9 protein which are correctly identified by enzyme digestion into the cytoplasm of the fertilized egg together;
6) microinjecting 300-400 fertilized eggs, and culturing the fertilized eggs after injection in an M16 (Sigma) culture medium in a 5% CO2 incubator at 37 ℃ for 1 hour;
7) preparing a 3-6 month-old female mouse for surrogate pregnancy one day before microinjection, injecting 120 mg/kg ketamine and 16 mg/kg xylazine into the abdominal cavity to anaesthetize the female mouse for surrogate pregnancy, and placing the female mouse on a constant temperature table;
8) shearing the skin of the abdominal cavity of the mouse by using sterile scissors, pulling out the uterine horn of the pregnant female mouse, maintaining the humidity by using 0.9 percent NaCl solution, and fixing the uterus;
9) clamping the upper end of ampulla of the fallopian tube by using forceps to form a small hole on the wall of the fallopian tube; injecting 20-30 fertilized eggs into the oviduct of each surrogate female mouse by using a thin glass capillary;
10) putting the uterus into the mouse body again, and putting the mouse into a breeding cage for breeding after surgical suture;
11) after the mouse is born for 2-3 weeks, shearing ear numbers, shearing tails, extracting a genome, amplifying fragments by PCR, and performing sequencing identification to obtain a positive F0-generation mouse;
12) sexually mature positive F0 mice were mated with wild type C57BL/6J mice to obtain F1 mice, and progeny mice of about 2 weeks after birth were tailed to extract genomes and identified by PCR using primer sequence 3 and primer sequence 4.
5. The method for preparing the targeting vector and its genetically modified mouse for differentiating the two variable cleavers of Tbx1 according to claim 4, further comprising:
the mouse genotype identification process comprises the following steps:
1) shearing 0.1-0.2 cm of mouse tail into 1.5 ml of EP tube, adding 150. mu.l NaOH (50mM) lysis buffer;
2) ensuring that the lysate is over the sample, and incubating overnight in a constant-temperature water bath kettle at 65 ℃;
3) centrifuging at 12000 rpm for 2 min, and sucking the supernatant;
4) adding 20 mul Tris-HCl (1M, PH7.5), and fully mixing;
5) phenol: chloroform: adding 150 mul of mixed solution into isoamyl alcohol =25:24:1, and uniformly mixing by vortex oscillation;
6) centrifuging at 12000 rpm for 2 minutes, and sucking the supernatant into a new EP tube;
7) adding 1 ml of precooled absolute ethyl alcohol, fully and uniformly mixing, centrifuging at 4 ℃ and 12000 rpm for 10 minutes, and removing supernatant;
8) drying in an ultraclean bench, dissolving the precipitate with 100. mu.l of double distilled water, and taking 2. mu.l as a template for PCR identification.
6. The targeting vector for differentiating two variable cleavers of Tbx1 and the method for preparing the genetically modified mouse thereof prepared according to claim 5, wherein the targeting vector comprises:
the positive mice were designated TMV (Tbx 1201 flox-V5-G15) and TMF (Tbx 1203 flox-3 xFlag-C9), respectively, as judged by F0 mouse PCR products:
1) the TMV positive mouse contains 3.0 kb and 4.1 kb fragments after being amplified by TMV-1 in the primer sequence 1, and the size of a product after TMV-2 amplification is 6.2 kb; only 4.1 kb fragment is obtained after TMV-1 amplification, and no fragment obtained after TMV-2 amplification is a negative mouse;
2) TMF positive mice amplify a 4.9 kb fragment through TMF-1 in a primer sequence 2, TMF-2 amplifies a 4.8 kb fragment, and negative mice amplify no fragments; the primer sequences for PCR amplification of F0 mouse are as follows:
primer sequence 1
TMV-1F：5’ -ACAGGCGGTGCTTGTCTTAG-3’
TMV-1R：5’ -AAGAGAGGCGATGCTGAACG-3’
TMV-2F：5’ -AGCATCGCCTCTCTTAAGTCC-3’
TMV-2R：5’ -GCCTCTGATGGGGAGTTTCC-3’
Primer sequence 2
TMF-1F：5’ -GCAAGATTTGCAGCTTATTAGCC-3’
TMF-1R：5’ -GTGGATTCGGACCAGTCTGA-3’
TMF-2F：5’ -ACGTAAACGGCCACAAGTTC-3’
TMF-2R：5’ -TGGATTTGGCGTCACTAGCCAG-3’。
7. The targeting vector and the method for preparing the genetically modified mouse thereof for differentiating two variable cleavers of Tbx1 according to claim 6, further comprising:
the sequences of primers used for PCR amplification of progeny mice are as follows:
primer sequence 3:
TMV-3F：5’ -CGTTCAGCATCGCCTCTCTT-3’
TMV-3R：5’ -GCCTGACAGTATAGACGCGG-3’
primer sequence 4:
TMF-3F：5’ -TGAAAAGCGGATGAAGGTGCAG-3’
TMF-3R：5’ -GGAAAATGAGCGCAATGGCTTTTA-3’。
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