CN115029382B - Preparation method of genetically modified mice capable of distinguishing Tbx1 two variable cutters - Google Patents
Preparation method of genetically modified mice capable of distinguishing Tbx1 two variable cutters Download PDFInfo
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Abstract
The invention provides a targeting vector capable of distinguishing Tbx1 variable cutter and a preparation method of a genetically modified mouse thereof, comprising the steps of designing and constructing the targeting vector of Tbx1 variable cutter 201 and Tbx 203, selecting intron I of Tbx1201 and Tbx 203 as an editing site, and co-injecting the targeting vector, guide RNA and Cas9 protein into fertilized eggs of the mouse through microinjection. The genetically modified mice of the invention can accurately distinguish two types of cutter of Tbx1, and the development of heart outflow tracts of mice homozygous for Tbx1203 mutation has obvious defects, thus providing a reliable animal model for revealing the action mechanism of the two types of variable cutter of Tbx1 in the occurrence process of congenital heart diseases.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a targeting vector capable of distinguishing Tbx1 two variable shear bodies and a preparation method of a genetically modified mouse.
Background
Early heart developmental defects are the leading cause of early embryonic death, accounting for 30% of cases of human embryo and early fetal death. Research on congenital heart disease pathogenesis models provides a guarantee for solving increasingly complex biological problems, and simultaneously avoids partial problems of ethical moral and technical limitations. Mice are well suited as animal models of congenital heart disease pathogenesis because the heart development process of mice is highly similar to that of human embryos, they have a four-chamber structure like humans, and have strong reproductive capacity, short reproductive cycle, stable genetic background, and mature operating system, and thus have 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 (22q11 deletion syndrome,22q11.2DS) using a mouse model of Tbx1 gene knockout. Specifically, heart outflow tract (OFT) was observed to exhibit mild defects in Tbx1 +/-mice, tbx 1-/-mice exhibited more severe heart, craniofacial, thymus and parathyroid defects, and the associated phenotype was consistent with the DiGeorge syndrome (DiGeorge syndrome, DGS)/palatofacial syndrome (velocardiofacial syndrome, VCFS) patient. In addition, mouse gene dose studies indicate that Tbx1 belongs to a dose-dependent gene, and the expression of Tbx1 in different tissues has differences in space and time, and the expression of Tbx1 needs to be precisely regulated in the embryo heart development process. Notably, the Tbx1 knockout mice reported are common region knockout to variable sham, and cannot accurately distinguish the role and mechanism of different variable sham in the developmental process. In other words, the Tbx1 gene knockout mice reported at present can prove the main functions of the gene and the regulation network thereof, but cannot distinguish the roles and mechanisms of different variable sheared bodies of Tbx1 in the heart development process. Therefore, there is a need for a mouse model that can accurately distinguish between studies of different cutter Tbx1, providing an effective tool for revealing the pathogenic mechanisms of different cutter.
Compared with Zinc Finger Nuclease (ZFNs) and transcription activation-like effector nuclease (transcription activator-like effector nuclease, TALEN) technologies, the CRISPR/Cas9 gene editing technology has the advantages of short period, high efficiency and strong specificity. Meanwhile, expression forms of different variable shears of Tbx1 are accurately distinguished by virtue of different tag proteins of V5 and 3xFlag, the deletion of corresponding shears is realized by point mutation, the conditional knockout of Tbx1201 and Tbx1203 can be realized by a Cre-loxp system, and the acquisition of related mice has important significance for revealing the 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 Tbx1 two variable cutters and a preparation method of a genetically modified mouse.
The invention provides a preparation method of a genetically modified mouse capable of distinguishing two variable shear bodies of Tbx1, which is characterized by comprising the following steps of:
designing and constructing targeting vectors of Tbx1201 and 203 variable cutter;
the Tbx1201 and 203 intron one was selected as the editing site;
the targeting vector, guide RNA and Cas9 protein were co-injected into mouse fertilized eggs by microinjection.
Furthermore, the targeting vector for distinguishing the Tbx1 variable cutter and the preparation method of the genetically modified mouse also have the following characteristics: the genetically modified mice were screened for offspring positive mice by PCR and gene sequencing methods.
Furthermore, the targeting vector for distinguishing the Tbx1 variable cutter and the preparation method of the genetically modified mouse also have the following characteristics: guide RNA sequence 1 matched to Tbx1 variable cleavage was: CTTTCGGTCAAGGACTTAGTAGG; guide RNA sequence 2 matched to Tbx1 variable cleavage was: CTGGGACTCTTCGAATTCGAGGG
Furthermore, the targeting vector for distinguishing the Tbx1 variable cutter and the preparation method of the genetically modified mouse also have the following characteristics:
embryo isolation and microinjection procedure:
1) On the first day, selecting 3-4 week old C57BL/6J female mice, wherein each mouse is intraperitoneally injected with 5IU of pregnant horse serum gonadotropin (PMSG), 5IU of Human Chorionic Gonadotropin (HCG) is intraperitoneally injected after 48 hours, and the mice are immediately mated with 2-8 months of male mice after injection;
2) The following day, the vaginal pessaries of the mice were checked before 9 am, and the donor female mice were separated from the male mice;
3) On the fourth day, fertilized eggs were collected from oviducts of female mice, and cumulus cells were placed in M2 medium containing 0.1% bovine hyaluronidase. Releasing fertilized egg, placing at 37deg.C, 5% CO 2 Culturing in an incubator;
4) Under an inverted microscope, selecting fertilized eggs with normal morphology and clear zona pellucida and male prokaryotes for microinjection;
5) Sucking the fertilized egg with a holding capillary, and co-injecting the enzyme-digested identified correct targeting vector, guide RNA and Cas9 protein into cytoplasm of the fertilized egg;
6) Microinjection of 300-400 fertilized eggs, placing the fertilized eggs after injection in M16 (Sigma) medium, and at 37 ℃ and 5% CO 2 Culturing in an incubator for 1 hour;
7) Preparing a surrogate female mouse of 3-6 months old day before microinjection, injecting 120mg/kg ketamine and 16mg/kg xylazine into the abdominal cavity to anesthetize the surrogate female mouse, and placing the surrogate female mouse on a constant temperature table;
8) Cutting the skin of the abdominal cavity of the mouse by using sterile scissors, pulling out the uterine horn of the female pregnant mouse, keeping the humidity by using 0.9% NaCl solution, and fixing the uterus;
9) The upper end of the ampulla of the oviduct is clamped by forceps, and a small hole is formed on the wall of the oviduct. Injecting 20-30 fertilized eggs into oviducts of each female-pregnant rat by using a fine glass capillary;
10 Placing uterus into the body of the mice again, and placing the mice into a rearing cage for rearing after surgical suturing;
11 After 2-3 weeks of birth, ear numbering is carried out, genome is extracted after tail cutting, PCR (polymerase chain reaction) amplification fragment and gene sequencing identification are carried out, thus obtaining positive F0-generation mice;
12 Sex mature positive F0 generation mice are respectively mated with wild C57BL/6J mice to obtain F1 generation mice, offspring mice with the age of about 2 weeks after birth are subjected to tail cutting, genome is extracted, and PCR identification is carried out by using a primer sequence 3 and a primer sequence 4.
Furthermore, the targeting vector for distinguishing two variable shear bodies of Tbx1 and the preparation method of the genetically modified mouse also have the characteristics that the targeting vector further comprises the following steps:
identification process of mouse genotype:
1) The tail of 0.1-0.2cm mice was cut into 1.5ml EP tubes and 150. Mu.l NaOH (50 mM) lysis buffer was added;
2) Ensuring that the lysate is not over the sample, and incubating the sample in a constant-temperature water bath at 65 ℃ for overnight;
3) Centrifuging at 12000rpm for 2 min, and sucking the supernatant;
4) Adding 20 μl Tris-HCl (1M, PH7.5), and mixing thoroughly;
5) Phenol: chloroform: 150 μl of the mixed solution is added in the ratio of isoamyl alcohol=25:24:1, and the mixture is uniformly mixed by vortex oscillation;
6) Centrifuge at 12000rpm for 2 min, aspirate supernatant into a new EP tube;
7) Adding 1ml of precooled absolute ethyl alcohol, fully and uniformly mixing, centrifuging at a temperature of 4 ℃ and at a speed of 12000rpm for 10 minutes, and discarding the supernatant;
8) Dried in an ultra clean bench, the pellet was dissolved in 100. Mu.l double distilled water, and 2. Mu.l was used as a template for PCR identification.
Furthermore, the targeting vector for distinguishing two variable shear bodies of Tbx1 and the preparation method of the genetically modified mouse also have the characteristics that the targeting vector further comprises the following steps:
f0 generation mice PCR product determination, positive mice were named TMV (Tbx 1201 flox-V5-G15) and TMF (Tbx 1203 flox-3 xFlag-C9), respectively:
1) TMV positive mice contained 3.0kb and 4.1kb fragments after amplification by TMV-1 in primer sequence 1, and the size of the amplified TMV-2 product was 6.2kb. TMV-1 amplified was only 4.1kb, TMV-2 amplified no fragment was negative mice.
2) TMF positive mice amplified a 4.9kb fragment by TMF-1 in primer sequence 2, TMF-2 amplified a 4.8kb fragment, while negative mice amplified no fragment.
The primer sequences used for PCR amplification of F0 mice 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’
Furthermore, the preparation method of the targeting vector for distinguishing the Tbx1 variable cutter and the genetically modified mice is characterized by further comprising the following steps:
determination of the size of the offspring mouse PCR product:
1) In the PCR product of TMV mouse genome primer sequence 3, the fragment size is 223bp only as homozygote mouse, at the same time, 181bp fragment is heterozygote mouse, and the rest is wild mouse.
2) In the PCR product of TMF mouse genome primer sequence 4, the fragment size is only 360bp of homozygous mice, at the same time 298bp fragment is heterozygote mice, and the rest is wild type mice.
The primer sequences used for PCR amplification of the offspring mice were 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
FIG. 1 shows the genomic positions of Tbx1201 and Tbx1203 cutter loci.
FIGS. 2A and 2B are schematic diagrams of construction strategies related to TMV and TMF gene modified mice.
FIGS. 3A and 3B are graphs of homologous recombination donor vectors of TMV and TMF gene modified mice.
FIGS. 4A and 4B show the results of the restriction enzyme digestion and identification of the homologous recombination donor vector of TMV and TMF gene modified mice.
FIGS. 5A and 5B are PCR identification electrophoretograms of F0 generation TMV and TMF gene modified mice 5 'homology arm and 3' homology arm.
FIGS. 6A and 6B are the identification of F1 generation TMV and TMF mouse PCR products.
FIGS. 7A and 7B are the identification of F2 generation TMV and TMF mouse PCR products.
Fig. 8 is a heart elevation view of an F2 generation TMF, TMV homozygous mouse.
Detailed Description
The technical scheme of the invention is further described below with reference to the specific embodiments.
According to the invention, a homologous arm is designed to construct a targeting vector by selecting a Tbx1 gene sequence in a mouse chromosome 16, V5 and 3xFlag tag proteins are added near the start codons ATG of Tbx1201 and 203, and point mutations are introduced into an exon of the variable cutter to respectively knock out Tbx1201 and 203. Adding flox within intron number one containing Tbx1201 and 203 can achieve co-knockout of Tbx1201 and 203 by breeding with Cre expressing mice. Editing mice are obtained through CRISPR/Cas9 technology, intron number one of variable cutter is selected as editing site, guide RNA is transcribed in vitro, targeting vector, guide RNA and Cas9 protein are injected into fertilized eggs of the mice together by microinjection, and positive mice are screened from offspring through PCR and gene sequencing methods. The generated genetically modified mice carry V5 and 3xFlag tag proteins and can be identified by corresponding antibodies, so that an effective strategy and scheme are provided for solving the problem that the current Tbx1 antibody cannot distinguish Tbx1 variable cutter types.
1. Genetically modified sites and guide RNAs
1. Gene modification site
1.1 mouse Tbx1 Gene (GenBank accession number: NM-001285472.1, ensembl database number: 00000009097) is located on mouse chromosome 16.
1.2Tbx1 locus has 5 variable cutter.
1.3Tbx1201 variable cutter (ENSMUST 00000009241.7) contains a total of 9 exons and Tbx1203 variable cutter (ENSMUST 00000232335.2) contains a total of 7 exons.
1.4 this modification adds a flox sequence within intron number one of the variable cutter of Tbx1201 and 203. The V5 tag protein is added near the start codon ATG of the variable cutter of Tbx 1201. The 15 th base is removed after the start codon ATG of the variable cutter of Tbx1203 to cause point mutation.
1.5 this modification adds a flox sequence to intron one of Tbx1201 and 203 variable cutter, tbx1203 variable cutter initiation codon ATG followed by 3xFlag tag protein, and removal of the C base at position 9 after Tbx1201 variable cutter initiation codon ATG resulted in point mutation.
1.6 this modification will design a targeting vector, the homology arm was obtained by PCR amplification using BAC clone, RP24-78F18 as template.
1.7 the TMV targeting vector modified at this time is identified by SacI restriction enzyme digestion, and the TMF targeting vector is identified by Asc1 and 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 selected as editing sites the two variable cutter intron numbers one of Tbx1201 and Tbx 1203.
1.10Cas9 protein and in vitro transcribed guide RNA were co-injected with targeting vector into mouse fertilized eggs.
1.11 offspring mice born will be screened for positive mice by genotyping analysis by PCR and gene sequencing methods.
2. Genetic modification
2.1 schematic diagram
The positions of the different variable cleavages of Tbx1 on the genome are shown in FIG. 1, the exons are arranged from left to right, the Tbx1201 variable cutter is about 8.9kb in length and the Tbx1203 variable cutter is about 5.3kb in length, the open reading frame ORF being shown in solid bars in FIG. 1.
2.2Guide RNA sequences
Guide RNA sequence 1 matched to Tbx1 variable cleavage was: CTTTCGGTCAAGGAC TTAGTAGG
Guide RNA sequence 2 matched to Tbx1 variable cleavage was: CTGGGACTCTTCGAA TTCGAGGG
2. Embryo separation and microinjection procedure
1) On the first day, selecting 3-4 week old C57BL/6J female mice, wherein each mouse is intraperitoneally injected with 5IU of pregnant horse serum gonadotropin (PMSG), 5IU of Human Chorionic Gonadotropin (HCG) is intraperitoneally injected after 48 hours, and the mice are immediately mated with 2-8 months of male mice after injection;
2) The following day, the vaginal pessaries of the mice were checked before 9 am, and the donor female mice were separated from the male mice;
3) On the fourth day, fertilized eggs were collected from oviducts of female mice, and cumulus cells were placed in M2 medium containing 0.1% bovine hyaluronidase. Releasing fertilized egg, placing at 37deg.C, 5% CO 2 Culturing in an incubator;
4) Under an inverted microscope, selecting fertilized eggs with normal morphology and clear zona pellucida and male prokaryotes for microinjection;
5) Sucking the fertilized egg with a holding capillary, and co-injecting the enzyme-digested identified correct targeting vector, guide RNA and Cas9 protein into cytoplasm of the fertilized egg;
6) Microinjection of 300-400 fertilized eggs, placing the fertilized eggs after injection in M16 (Sigma) medium, and at 37 ℃ and 5% CO 2 Culturing in an incubator for 1 hour;
7) Preparing a surrogate female mouse of 3-6 months old day before microinjection, injecting 120mg/kg ketamine and 16mg/kg xylazine into the abdominal cavity to anesthetize the surrogate female mouse, and placing the surrogate female mouse on a constant temperature table;
8) Cutting the skin of the abdominal cavity of the mouse by using sterile scissors, pulling out the uterine horn of the female pregnant mouse, keeping the humidity by using 0.9% NaCl solution, and fixing the uterus;
9) The upper end of the ampulla of the oviduct is clamped by forceps, and a small hole is formed on the wall of the oviduct. Injecting 20-30 fertilized eggs into oviducts of each female-pregnant rat by using a fine glass capillary;
10 Placing uterus into the body of the mice again, and placing the mice into a rearing cage for rearing after surgical suturing;
11 After 2-3 weeks of birth, ear numbering is carried out, genome is extracted after tail cutting, PCR (polymerase chain reaction) amplification fragment and gene sequencing identification are carried out, thus obtaining positive F0-generation mice;
12 The sexual mature positive F0 mice are respectively mated with wild C57BL/6J mice to obtain F1 mice, the offspring mice with about 2 weeks age after birth are cut off, the genome is extracted, and the PCR identification is carried out by using a primer sequence 3 and a primer sequence 4.
3. Mouse genotyping
1. Sample treatment:
1) A0.1-0.2 cm mouse tail was cut into a 1.5ml EP tube and 150. Mu.l NaOH (50 mM) lysis buffer was added.
2) Ensure that the lysate is submerged in the sample and incubated overnight in a 65℃thermostat water bath.
3) Centrifuge at 12000rpm for 2 min and aspirate the supernatant.
4) Mu.l of Tris-HCl (1M, PH7.5) was added and thoroughly mixed.
5) Phenol: chloroform: isoamyl alcohol=25:24:1 was added to 150 μl of the mixture, and mixed by vortexing.
6) Centrifuge at 12000rpm for 2 min and aspirate the supernatant into a new EP tube.
7) 1ml of pre-chilled absolute ethanol was added, thoroughly mixed, centrifuged at 12000rpm for 10 minutes at 4℃and the supernatant was discarded.
8) Dried in an ultra clean bench, the pellet was dissolved in 100. Mu.l double distilled water, and 2. Mu.l was used as a template for PCR identification.
Restriction enzyme identification of Donor vector
As shown by the result of the SacI digestion identification of the TMV Donor vector in FIG. 4A, the sizes of fragments after digestion are 8.5kb, 4.0kb, 1.4kb and 0.8kb respectively, which are expected, and the construction of the TMV Donor vector is proved to be successful. From FIG. 4B, a 10.5kb fragment was obtained after cleavage of TMF Donor vector by Asc1, and the fragment sizes after cleavage of Mfe1 and Nhe1 were 6.3kb, 2.2kb, 1.4kb and 0.7kb, which were expected to prove the success of TMF Donor vector construction.
CRISPR induced modification detection
Target areas of different variable cutters of the mouse Tbx1 are amplified by PCR through specific primers, and sequencing verification is carried out on amplified products, so that the correctness of genome editing positions is ensured.
The primer sequences used for PCR amplification of F0 mice 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’
The primer sequences used for PCR amplification of the offspring mice were 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’
PCR amplification product size:
TMV wild-type gene use: primer sequence 3, product length is 181bp.
TMV heterozygous gene use: primer sequence 3, product length is 181bp and 223bp.
TMV homozygous gene use: primer sequence 3, product length was 223bp.
TMV wild-type gene use: primer sequence 4, product length 298bp.
TMV heterozygous gene use: primer sequence 4, product length is 298bp and 360bp.
TMV homozygous gene use: primer sequence 4, product length is 360bp.
PCR products, as shown in FIGS. 5A and 5B.
As shown in FIG. 5A, the PCR products of the 5 '-arm of the mice of F0 generation 3, 4, 8 and 9 contained bands of 4.1kb and 3.0kb, and the PCR products of the 3' -arm contained bands of 6.2kb, which proved that the above mice were heterozygous mice.
As shown in FIG. 5B, the PCR products of the 5 '-arm of the mice of F0 generation 29, 31 and 39 simultaneously contain a 4.9kb sized band, and the PCR products of the 3' -arm contain a 6.2kb sized band, which proves that the mice are heterozygous mice.
From FIG. 6A, the PCR products of mice of the F1 generation, TMV mice, no. 3, 4, 8, 9 and 10, contain fragments of 223bp and 181bp, which prove that the mice are heterozygous mice.
From FIG. 6B, the PCR products of mice of the F1 generation, TMF mice, no. 1, 2, 3 and 4, contain fragments of 360bp and 298bp, which prove that the mice are heterozygous mice.
From FIG. 7A, the PCR products of mice of the F2 generation contain fragments of 223bp and 181bp, which prove that the mice are heterozygous mice, as shown by the detection results of the TMV mice of the F2 generation; mouse # 12 contained only the 223bp fragment as homozygous mice, with the remainder being wild type mice.
From FIG. 7B, the PCR products of mice of the F2 generation contain fragments of 360bp and 298bp, which prove that the mice are heterozygous mice. Mouse No. 7 contained only 360bp of the fragment as homozygous mice, the remainder of the mice No. 5 being wild type mice.
As can be seen from fig. 8, the outflow tract of the TMF homozygous mice (left in the figure) of the first day (P0) of birth is divided into the aorta and the pulmonary artery, with no obvious abnormality; the aorta and pulmonary artery of the TMV homozygous mice (right panel) of day one (P0) were fused, and the cardiac outflow tract was markedly abnormal.
Claims (1)
1. A method for preparing a genetically modified mouse capable of distinguishing between two variable segmentes Tbx1, comprising designing and constructing targeting vectors for the variable segmentes Tbx1201 and 203;
the first intron of Tbx1201 and 203 was selected as the editing site;
co-injecting the targeting vector, guide RNA and Cas9 protein into the fertilized eggs of the mice by microinjection;
the targeting vector is prepared by cloning an exogenous nucleic acid construct into a mouse genome by means of homologous recombination based on a CRISPR/Cas9 system and a Cre/LoxP system; the targeting vector comprises a tag protein V5 or 3xflag, mutated Tbx1201 or Tbx1 203; the Tbx1201 variable cutter is ENSMUST00000009241.7, the Tbx1203 variable cutter is ENSMUST00000232335.2, the mutations are a point mutation caused by removing a C base at position 9 after the Tbx1201 variable cutter start codon ATG and a point mutation caused by removing a G base at position 15 after the Tbx1203 variable cutter start codon ATG, the mutated Tbx1201 variable cutter start codon ATG is followed by the V5 tag protein coding sequence, and the mutated Tbx1203 variable cutter start codon ATG is followed by the 3xFlag tag protein coding sequence;
the genetically modified mice are screened for offspring positive mice by PCR and gene sequencing methods;
the sequences matched with the Tbx1 variable cleavage phase comprise a guide RNA sequence 1 and a guide RNA sequence 2, wherein the guide RNA sequence 1 is as follows: CTTTCGGTCAAGGACTTAGTAGG; the guide RNA sequence 2 is as follows: CTGGGACTCTTCGAATTCGAGGG.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109112162A (en) * | 2018-08-30 | 2019-01-01 | 西南大学 | Mouse model and construction method are knocked in situ using the Huntington disease that CRISPR/Cas9 technology constructs |
| CN111304259A (en) * | 2020-02-24 | 2020-06-19 | 内蒙古自治区人民医院 | Method for constructing ABCC4 gene knockout mouse by CRISPR/Cas9 technology and application thereof |
| CN111485002A (en) * | 2020-05-06 | 2020-08-04 | 澎立生物医药技术（上海）有限公司 | Preparation method and application of transgenic mouse with severe immunodeficiency |
| CN113558011A (en) * | 2021-07-23 | 2021-10-29 | 深圳市中医院 | Method for establishing sjogren syndrome animal model based on gamma-secretase activated protein gene |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109266680B (en) * | 2018-10-17 | 2020-09-25 | 江苏集萃药康生物科技有限公司 | Method for preparing CKO/KI animal model by using Cas9 technology |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109112162A (en) * | 2018-08-30 | 2019-01-01 | 西南大学 | Mouse model and construction method are knocked in situ using the Huntington disease that CRISPR/Cas9 technology constructs |
| CN111304259A (en) * | 2020-02-24 | 2020-06-19 | 内蒙古自治区人民医院 | Method for constructing ABCC4 gene knockout mouse by CRISPR/Cas9 technology and application thereof |
| CN111485002A (en) * | 2020-05-06 | 2020-08-04 | 澎立生物医药技术（上海）有限公司 | Preparation method and application of transgenic mouse with severe immunodeficiency |
| CN113558011A (en) * | 2021-07-23 | 2021-10-29 | 深圳市中医院 | Method for establishing sjogren syndrome animal model based on gamma-secretase activated protein gene |
Non-Patent Citations (3)
| Title |
|---|
| "Chromatin and Transcriptional Response to Loss of TBX1 in Early Differentiation of Mouse Cells";Andrea Cirino et al.;《Frontiers in Cell and Developmental Biology》;第8卷;第1-14页 * |
| "Single cell multi-omic analysis identifi es a Tbx1- dependent multilineage primed population in murine cardiopharyngeal mesoderm";Hiroko Nomaru et al.;《NATURE COMMUNICATIONS》;第12卷;第1-19页 * |
| "TBX1 基因影响 22q11.2 微缺失综合征表型的作用机制研究进展";路玥 等;《医学研究生学报》;第33卷(第6期);第664-668页 * |
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