CN118202978B - Stella-CreERT2 animal model and construction method and application thereof - Google Patents

Stella-CreERT2 animal model and construction method and application thereof Download PDF

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CN118202978B
CN118202978B CN202410493014.5A CN202410493014A CN118202978B CN 118202978 B CN118202978 B CN 118202978B CN 202410493014 A CN202410493014 A CN 202410493014A CN 118202978 B CN118202978 B CN 118202978B
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pgc
stilla
creert
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mouse
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CN118202978A (en
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陈子江
赵世斗
秦莹莹
杨亚娟
许伟伟
温灿鑫
陈才艺
丛洪斌
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Shandong University
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Abstract

The invention provides a stilla-CreERT 2 animal model, and a construction method and application thereof. In the invention, an inducible and tissue-specific stilla-CreERT 2 animal model is successfully constructed by means of CRISPR-Cas9 technology and a CreERT/loxP gene recombination system; and constructing and obtaining Mcm8 fl/fl of the specific knockout mini chromosome maintenance coding gene 8 in the Primordial Germ Cells (PGCs) from the construction; the stilla-CreERT 2 mice were first found to knock out Mcm8 gene in PGCs, resulting in a significant decrease in embryo PGC numbers. The invention provides a preparation method of an inducible conditional gene knockout tool mouse, which can effectively control the space-time specificity knockout of a target gene in PGC by a Creert/loxP system and provides a powerful tool for exploring the functions and mechanisms of key regulatory genes in PGC development.

Description

Stella-CreERT2 animal model and construction method and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a stilla-CreERT 2 animal model, and a construction method and application thereof.
Background
The statements in this section merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Primordial germ cells (Primordial germ cell, PGCs) are gamete precursor cells that are induced in the posterior of the proximal ectoderm of the early embryo of a mammal, and the number of PGCs produced by mitotic proliferation is a determinant of the establishment of reproductive reserves, whose normal development is critical for gametogenesis and species progression. Gene knockout mice can be an effective model for studying key regulatory factors in PGC development, but systemic knockout lacks cell specificity and may lead to embryonic lethality, limiting its use.
The Cre gene is inserted behind a promoter of a tissue specific endogenous gene, so that Cre enzyme is expressed in specific tissues, and the target gene with LoxP site in specific tissue cells can be knocked out. It has been reported that Blimp1-Cre, stilla-Cre, tnap-Cre and Oct4-Cre can achieve specific knockout of target genes in mouse PGCs. However, in addition to PGCs, blimp1 gene is expressed in various ectodermal-derived tissues (primitive retinal neurons, etc.), mesodermal-derived tissues (visceral wall layers, etc.), endodermal-derived tissues (foregut, midgut, hindgut endodermal epithelial cells, etc.), stilla gene is also expressed in early embryo before implantation, tnap gene is also expressed in placenta, intestine, neural tube, etc., oct4 is expressed in blastocyst-inner cell mass multipotent stem cells, and thus the above mice are prone to Cre enzyme organ leakage, which may affect PGC phenotype observation and mechanism research.
At present, a strategy for constructing a Cre mouse is usually to target through embryonic stem cell (Embryonic stem cell, ESC) genes, insert the Cre genes behind endogenous gene promoters, and although Cre enzyme expression driven by the endogenous gene promoters can be realized, the strategy destroys the expression of the endogenous genes, so that the problems of incapability of homozygosity, fertility reduction and the like of Cre tool mice are caused, and the proportion of genotype of interest in offspring mice mated with the floxed mice is low. In addition, the Cre enzyme in the mice exhibits activity after expression, and the target gene knockout time is substantially identical to the time at which the endogenous gene starts to be expressed, so that the target gene cannot be knocked out at regular intervals.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a stilla-CreERT 2 animal model, and a construction method and application thereof. In the invention, an inducible and tissue-specific stilla-CreERT 2 animal model is successfully constructed by means of CRISPR-Cas9 technology and a CreERT/loxP gene recombination system; and constructing and obtaining Mcm8 fl/fl of the specific knockout mini chromosome maintenance coding gene 8 in the PGC; the stilla-CreERT 2 mice were first found to knock out Mcm8 gene in PGCs, resulting in a significant reduction in PGC numbers in embryos. The method provided by the invention is simple, can effectively control the space-time specificity knockout of the target gene in the PGC by the CreERT/loxP system, and provides a powerful tool for exploring the functions and mechanisms of key regulatory genes in PGC development.
To achieve the above object, one or more embodiments of the present invention provide the following technical solutions:
in a first aspect of the present invention, there is provided a method of constructing a stilla-CreERT 2 animal model, the method comprising the steps of:
1) Constructing homologous recombinant plasmids;
2) Injecting Cas9 mRNA, sgRNA and homologous recombinant plasmid into fertilized eggs of animals through microinjection, and transplanting the fertilized eggs after injection into a gestation animal body to obtain F0-generation animals;
3) Mating the F0 generation animal with a wild type animal to obtain an F1 generation;
4) The F1 generation was identified as positive as a Stella-CreERT2 animal.
Wherein the model animal may be a mouse, rat, mouse, rabbit, dog, or monkey; preferably a mouse.
In a specific embodiment of the invention, the homologous recombinant plasmid comprises a 5 'homology arm, a P2A-CreERT2 fragment and a 3' homology arm which are connected in sequence;
The nucleic acid sequences of the 5 'homology arm, the P2A-CreERT2 fragment and the 3' homology arm are respectively shown in SEQ ID NO. 1-3.
SEQ ID NO.1:
cctggactagaactccctgtgcaatccagaatggcatcccaaatccgagaattaaagattaaaggcttccgagtgggctgcagtgtgtgtgtgtgtgggggggggcgttgtttgtgttgttttagcatagaaatcttggttgagacagtctcactctgtggtagtagatacaaggaacgcaaccttgtagccctggctgggctcagtctcgatcctcctgcctctgcctccaaagttttagaattaaaagtgtgcagtgtcatgccggcccttgtgtagcaccggagttttgtttgcttctctgtgtagcccaggttgtcctaggacgctgtacaccaggctggcttgggactcaggaggatccgcctttctcagactttcgagtgcagggattaaaggcagtgccacctcggtgggcgacagcggaatcttttaagcttcagttgaacctgggtttttttttttttttttttttttttttttcttattctcatctactttctttccagctaaatatccttttcaggtaaatggctattcaaggggagcccaagataggctgagccgtgtgctacatgggcccactggttcccactccccagctgcagttgattctttgtccgagggactgcctccgggcactcccggtctagggcgtggctcgagaggagccctcccctccggtctgattagaaaacaaaaaaatgccttgaatctgccagttacacgggaccttcgtatttccacgagataattctttagcctttgggggctttttaaatcttgaggctgttcagcccctactggtctaaaacccactatccaagctaggctagatccaaactctcaatcacttcggaggcagaaacgctggctggctggctcagccttggagtcactttgcctttcttccaggagcgttgtggttacgttttctgtataagatggggccatctactgcgtgtggacgattcaaacggagagagctggtttcccaacatttgtttttcgagactgttttcaagacagggtttcttctctgtgtagccccaggctgacctcgaactcagaaacccgcctgcctctgcctcccaagtacagggattaaaggcgtgcgccaccactgtctggcaacatcccttgttacatagctgtccagaagcatttgagcaggtcagttagatttaggtggaaaaatgaacagccagttttgggaaggttttccagagctgaagctgaacccagaggcactattggggaagccctccagctgagccacattcctgcagcacaattggctgcagagtctatgggagaaggggggggggggggagaatatgcaggtctcagaacctctgaactagacttacatacaggctgcatcggtaacccacagtaaagtagcgcagtggatctctacacaagcactagggtttatgactaagtcctgtgcacgatttgcttcttgttgacccctctgcggaagagggagtatccacttgtttttctctggggtgtattgtctttatgcctcactagtgtcttctgtttcagACGTCCTACAACCAGAAACACTAGTAAAGGTCATGAAAAAGCTAACCCTAAACCCCGGTGTCAAGCGGTCCGCACGCCGGCGCAGTCTACGGAACCGCATTGCAGCCGTACCTGTGGAGAACAAGAGTGAAAAAATCCGGAGGGAAGTTCAAAGCGCCTTTCCCAAGAGAAGGGTCCGCACTTTGTTGTCGGTGCTGAAAGACCCTATAGCAAAGATGAGAAGACTTGTTCGGgtgagtttcctgtgcggacagggctgttagacctaagagcaactccagcctcaacgggattcaaggcattttttgttttctaatcagaccggagtatagacaaggctagtgactgcttgtaattcgcagacggagagaatagagttccaaggctatcctctaagcagtcaggctgacctgagctatgtacatatacgcagtaatgagcatcagcactgggtgtttgaaagttaagactccctccctgtccaagctggccctccagttcactccacagtcctgcctcttcagagctgggactacagggctaacttagactagatctcagagctgaagtgggcctgttactctttgtaaaccaggctggccttaaactggggtgatcttcctgcccctgctttcacagagcctagcagtgacaacttctcacaaagtgtttaaagtaggtcttgatagttttgaataattttgtttttgtttttcttttaaagATTGAGCAGAGACAAAAAAGGCTCGAAGGAAATGAGgtaaatactctttgtttttgttttttgttttgtcttgctttcttttattttattttgtttggagacagggtttctctgtataaccctggctgtcctggaactcactgtagaccaggctggcctcgaactcagaaatccacctgcctctccctcccaagtgctaagattaaaggcatgtgccaccagaggtaaatactctttaaaatttttattatgagtacactgtagctgtcttcagacacaccagaagagggcgtcagatctcgttacggatggttgtgagccaccatgtggttgctgggatttgaactcaggacctttggaagagcagtcagtgctcttaacctctaagccatcactccagtcccaataaatactcttaatgcatgggtgggttgggtgtctgtttcattgtttgatttagactaggaatgtgtctggggagtatgtggaaaaaattctcagcccccaggaagtctggttattgaagcagtaattaaaacaatagataaatgggatttgtttcattgggtttacaatatcctgattaacctattttgttatttatttcagTTTGAACGGGACAGTGAGCCATTCAGATGTCTCTGCACTTTCTGCCATTATCAAAGATGGGATCCCTCTGAGAATGCGAAAATCGGGAAGAAT
SEQ ID NO.2:
GGTAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGCGACGTGGAGGAGAACCCTGGTCCTATGGGCTCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGGATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGATCTCGAGCCATCTGCTGGAGACATGAGAGCTGCCAACCTTTGGCCAAGCCCGCTCATGATCAAACGCTCTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTCAGTGCCTTGTTGGATGCTGAGCCCCCCATACTCTATTCCGAGTATGATCCTACCAGACCCTTCAGTGAAGCTTCGATGATGGGCTTACTGACCAACCTGGCAGACAGGGAGCTGGTTCACATGATCAACTGGGCGAAGAGGGTGCCAGGCTTTGTGGATTTGACCCTCCATGATCAGGTCCACCTTCTAGAATGTGCCTGGCTAGAGATCCTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCACCCAGTGAAGCTACTGTTTGCTCCTAACTTGCTCTTGGACAGGAACCAGGGAAAATGTGTAGAGGGCATGGTGGAGATCTTCGACATGCTGCTGGCTACATCATCTCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGTTTGTGTGCCTCAAATCTATTATTTTGCTTAATTCTGGAGTGTACACATTTCTGTCCAGCACCCTGAAGTCTCTGGAAGAGAAGGACCATATCCACCGAGTCCTGGACAAGATCACAGACACTTTGATCCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCGGCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCAGGCACATGAGTAACAAAGGCATGGAGCATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGACCTGCTGCTGGAGGCGGCGGACGCCCACCGCCTACATGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAGGAGACGGACCAAAGCCACTTGGCCACTGCGGGCTCTACTTCATCGCATTCCTTGCAAAAGTATTACATCACGGGGGAGGCAGAGGGTTTCCCTGCCACAGCTTGATGA
SEQ ID NO.3:
GAGCTTACATTGTACGCTGCCCTGGCTGTCGACGATGCCGCACAGCAGATGTGAAAGCTATTTTTTGTTTAAGATTAAACTTTTTCTGGTGCTGGGAAATCTTAACTTGTTAACCTTTAAATTGTAGATAGGATGCACAACGATCCAGATTTATGTGAAGTTTAGAAGCCTCAAGCTGTGAGGCCCAGGGCTGAGGAATAAAGTAAATAGAATTTGGAGTATGTACGTTCTAATTTCCAGAAATTTGTAATAAAAGCATTTTTGTTAgctcgactctttgtaatttacacaaacagctaggggctactgtaacagttctagaaatataaaattgtacattaagtaggaaaagacaaaatctcttactgtgcctaaattagaactttgacaataggtataatgtgttggctaggggtaaagcttgctgccaggcctggcagagacccacatgttggaaacatcttgcaagttgtcttctgacttccacatgagcatcttgacatgccaaaatctttagttccagcatttggaaggcagagacaggtggatctctgaccagtctggtttatagcccaggacagccagggctatgagtccctgtctcactttttaaaactagttccaggttctaggtgatctggaggtctattgagtcaagcactaattggcaggttatttgtttgcctcaacccctttcattagtgtgaaggtgctgaccaccaatgcccggcagagctgtagcccattatcacatttcctaggaggtttttgtttgtcttgagacagtatctccaggtttagctggcctggaattcacaagagagccacctgcctctgcctcccaagagctggtatcaaaggtttgtaccactatctctccaatccagaactcatgagatgttctaaatattttttaagttttaagactaggccaatgagcttttgtgtacattccacttacaccagttaatgacaatttaaaaaaattcttacatataaatacactgttgctgtcttcagacacaccagaagaggacatgggatcccattatagatgtttgtgagccaccatgtggttgctgggattttgaactcaggacctctgggagagcagtccgtgctcttagctgagccatctctgcagcccagtgttattcattttataaaatgttctgggtggttcactagaaaggaagctgagaaattatctgaatgttttgatccctagctacttgctgctctaccacttggaccatatgacccagcagacccgatggtacttgaggtatctgtggctgatagagatgctgtttggagcctctggcaggcccctgtaggtgaatcacagaaaagacctttgggatttttggagcaaagctctaccatcatctgcagacaactattctccctttgaaaaacagctcttggcctgctattgggccttagtggaaactgaacgtttgacaataggacaccaagttactatgcgacctgaactacccatcatgagctgggtactatcagaccctgcaagtcataaagtgggatgcgcacagcagcagtctattatcaaatggaagtggtatatacgtgatcgggccagagcaggtcctgaaggcacaagcaagttacatgaaaaagttgctcaaatgcctatggtttctactcctgttacaatgccatctgctgccaagcatgcgcctatagcctcatggggtgttccctatgatcgactgaccaaagaggagaagactagggcctggtttactgatggctctgcacgttatgcaggcaccacccagaagtggacagctgctacatgcttgccctccaatctccatatgtgtgtaatactacacagggttggataaccagtgtgggcagcatgacctctgccctgaaaggaagtgaaagccctcagtttcagggttctcttgcagttgtatattgcctccatatatataaaacctagcctttgtgttcaataaggtgagaaaaattagagttgcccttggagctaagtgtctgtgtcttgcctgcacactggtgtgcctcaggggacggctctgaggggttggttgctcctggatctcactgtcagggaactcttcccacagttacatgtgggcatctgttttctggccatgtcttcaggaaggatttgggtcagagtggcctgtgtgttctcagtaccgatgcctccctttagcttagagagaattcctcctcccctaagcagccttcaatgtctctccagtgctaagatgtaatctgtacatagtggatatgggacagtttgattgcacactaggcaaacactctactacctgagcagaatcccggactgcttcagctcattatttatacagattcgcctgtagccaaagctgccctactctctctagctgaggatctccaaatgctgggactcttggtttggcatgctactcccacctggtttttcagcttaaggtgcaaacaaattgcttccacgtggttatttttcatgcctttgctattcattaaagaaaccagagccgggcggtgaaggctctctatttagagaaattagtctaactatatctgttatgtaacatccattctagaccttctagttaatgagaaggcaggaaagagttaccagatacctgggaaatcaggtcctcttggagtgttcccccaaaatccacctctagacagggtttctttgtgtagccctgatcgtcctggaactcattctgtagaccaggctagccttgaatccaaggattcacctgctgctgcctctctagcactgggtttaaaggcgtagctcccaaagcccggcccttctcagagttcctagtttaaatcaaagaatttagggatggttacaaacatgtctaaggataatttattagagttaaaaacaaaactaggtatgctgcttgagacaaggtggcacaggttagcctcctggc
The homologous recombinant plasmid is obtained by an In-Fusion cloning method; the backbone vector of the homologous recombinant plasmid is pBR322.
In a specific embodiment of the invention, the target of the sgRNA is located on the 3' utr of the fourth exon of the stilla gene; ensembl number ENSMUST00000049644.9 of the stilla gene;
the sgRNA nucleic acid sequence is shown as SEQ ID NO. 4.
SEQ ID NO.4：GCTTACATTGTACGCTGCCCTGG。
In a specific embodiment of the invention, the animal fertilized egg is a C57BL/6J mouse fertilized egg;
Further, the fertilized eggs of the C57BL/6J mice are obtained after 0.5 day fertilization by hybridization of the C57BL/6J mice.
The identification criteria for positive mice were: the DNA of the mice to be identified is amplified by a primer I GGTATCTCGGTTGCGGGATT (SEQ ID NO. 5) and a primer II ATCCGGACAGTGGTTTCACC (SEQ ID NO. 6), a 5.2kb fragment is amplified by a 5' -arm homologous recombination positive genome, and a 6.7kb fragment is amplified by a negative genome; the DNA of the mice to be identified is amplified by primer III CCGAGCTAGCTTTTGAGGCT (SEQ ID NO. 7) and primer IV CTCTCCGCCTGGGTTTTCTT (SEQ ID NO. 8), the 3.5kb fragment should be amplified from the 3' -arm homologous recombination positive genome and the 7.0kb fragment should be amplified from the negative genome.
In a second aspect of the invention, there is provided a stilla-CreERT 2 animal model obtained by the method as above.
In a third aspect of the invention, there is provided a method of constructing a stilla-CreERT 2 animal model as above or the use of a stilla-CreERT 2 animal model in any one or more of the following:
1) Knocking out embryo PGC development related genes, preferably a minichromosome maintenance coding gene 8;
2) For modulating PGC development;
3) For constructing PGC dysplasia animal models;
4) Used for regulating embryo development;
5) For constructing an animal model of embryo dysplasia;
6) For PGC lineage tracing;
7) Is used for researching the action and mechanism of PGC development related genes.
According to a fourth aspect of the invention, a construction method of a Mcm8 gene knockout model mouse is provided, the stilla-CreERT 2 animal in the second aspect is hybridized with the Mcm8-flox mouse, and tamoxifen is injected into the abdominal cavity of the pregnant mouse to induce Mcm8 gene knockout to obtain Mcm8 gene knockout model mouse Mcm8 fl/fl in PGC; stella-CreERT2.
The induction concentration of tamoxifen is 0.07-0.08 mg/g/dose.
Mcm8-flox mice refer to mice in which two loxP sites are inserted at both ends of exon 4-5 of Mcm8-201 transcript, respectively.
In a fifth aspect of the present invention, there is provided a Mcm8 gene knockout model mouse obtained by a method of constructing a Mcm8 gene knockout model mouse.
In a sixth aspect of the present invention, there is provided a method of constructing a Mcm8 gene knockout model mouse according to the fourth aspect and/or use of a Mcm8 gene knockout model mouse according to the fifth aspect in any one or more of the following:
I) For modulating PGC development;
II) is used for constructing a PGC dysplasia mouse model;
III) for modulating embryo development;
IV) for constructing an embryonic dysplasia mouse model;
V) for PGC lineage tracing;
VI) is used for researching the action and mechanism of PGC development related genes.
In a seventh aspect of the invention, there is provided a kit for constructing a stilla-CreERT 2 animal model based on CRISPR-Cas9, the kit comprising Cas9 mRNA, sgRNA and homologous recombination vector as described in the first aspect;
wherein the Cas9 mRNA is obtained by in vitro transcription of a Cas9 nuclease.
The one or more of the above technical solutions have the following beneficial effects:
The invention applies CRISPR-Cas9 technology to efficiently construct the inducible stilla-Creert 2 tool mouse which can be stably inherited and specifically expressed. Mice PGC lineage tracing can be achieved by hybridizing the vehicle mice to reporter mice, and administering tamoxifen in combination. The tool mice are hybridized with target gene floxed mice, and tamoxifen is combined for administration, so that the target gene space-time specificity knockout mice in PGC can be obtained, and Cre enzyme is prevented from leaking in other tissues. The invention provides a powerful tool for exploring PGC development regulation mechanism, and has good application value.
The invention discovers that the Mcm8 gene is knocked out in PGC for the first time, so that the number of PGC in embryo is obviously reduced, and the Mcm8 gene is proved to be involved in regulating and controlling the development of PGC.
Additional aspects of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram of a mouse construction strategy according to an embodiment of the present invention.
FIG. 2 is a map of a homologous recombinant vector according to an embodiment of the present invention.
FIG. 3 is an electrophoresis chart of the identification of homologous recombination vector enzyme digestion according to the embodiment of the present invention.
FIG. 4 is a schematic diagram of the identification strategy of F0 mice in the first embodiment of the present invention.
FIG. 5 is a PCR identification electrophoresis chart of the genotype of a homologous recombination positive F0-generation mouse in the first embodiment of the invention.
Wherein Arabic numerals: f0 mice were numbered; WT: a wild-type control; m:1kb DNAmarker.
FIG. 6 is a PCR identification electrophoresis chart of the genotype of the homologous recombination positive F1-generation mice in the first embodiment of the invention.
Wherein Arabic numerals: f1 generation mice were numbered; WT: a wild-type control; m:1kb DNAmarker.
FIG. 7 shows the comparison result of the No. 3 mouse 1# sequencing reaction according to the embodiment of the present invention. The red underlined base is the 5' homology arm sequence.
FIG. 8 shows the comparison result of the No. 3 mouse 2# sequencing reaction according to the embodiment of the present invention. The red underlined base is the 5' homology arm sequence, and the blue underlined base is the knock-in sequence.
FIG. 9 is a comparison result of the 3# sequencing reaction of mouse # 3 in example one of the present invention. The blue underlined base is the knock-in sequence, and the red underlined base is the 3' -terminal homology arm sequence.
FIG. 10 shows the result of comparison of the 4# sequencing reaction of mouse # 3 in the example of the present invention. The red underlined base is the 3' homology arm sequence.
FIG. 11 is a diagram showing PCR identification of genotype of mice with subsequent offspring according to the first embodiment of the present invention.
Wherein HE: heterozygotes; WT: wild type; m:2000bp DNA marker.
FIG. 12 shows the results of E12.5 day mouse embryo STELLA protein fluorescent staining and TdTomato fluorescent protein detection in example two. Wherein, STELLA positive (green) shows PGC, GR shows genital ridge, and the statistical chart shows the proportion of STELLA +、TdTomato+ double positive cells to STELLA + cells.
FIG. 13 is a PCR identification electrophoresis chart of E12.5 day mouse embryo genotypes in the third embodiment of the present invention.
FIG. 14 shows the results of alkaline phosphatase staining of the embryo reproduction crest of E12.5 day mice in example III of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. Experimental methods in the following embodiments, unless specific conditions are noted, are generally in accordance with conventional methods and conditions of molecular biology within the skill of the art, and are fully explained in the literature. See, e.g., sambrook et al, molecular cloning: the techniques and conditions described in the handbook, or as recommended by the manufacturer.
The technical principle of the invention is as follows:
Aiming at the defects of the currently reported PGC specific Cre recombinase tool mice, such as damaged fertility of the Cre tool mice caused by the damage or interference of the normal functions of endogenous genes by the fusion protein of the modified gene Cre recombinase, incapacity of homozygously, low efficiency of knocking out the constructed Cre recombinase caused by the fact that the modified gene is expressed in tissues except PGC, and the like, the problems of multi-organ tissue leakage, reduced specificity, low efficiency of knocking out the constructed Cre recombinase and the like of the PGC specific Cre recombinase tool mice are solved. According to the invention, an inducible stilla-CreERT 2 animal model is successfully constructed by knocking in a P2A-CreERT2 expression sequence at a fixed point before a stilla gene stop codon through a CRISPR-Cas9 technology. When PGC expresses stilla, transcription of P2A-CreERT2 is started, and after translation is completed, P2A polypeptide is "self-cleaved" to respectively generate STELLA protein and CreERT2 fusion protein, so that influence on endogenous STELLA protein function is avoided. Therefore, the tool mice can be homozygous, and the proportion of the target genotype of the offspring mice is greatly improved by mating with the floxed mice. Furthermore, after tamoxifen is injected into pregnant mice, the metabolite 4-OHT of the pregnant mice is combined with Creet 2, so that Cre recombinase is promoted to enter cell nuclei to mediate homologous recombination among LoxP sites, and therefore, space-time specific knockout of target genes in target genotype mice PGC is realized. The invention provides a powerful tool for exploring the functions and mechanisms of key regulatory genes in PGC development.
Noun interpretation:
mcm8-flox mice: mice with two loxP sites inserted at both ends of exon 4-5 of Mcm8-201 transcript, respectively.
PGC lineage tracing: the development and differentiation of individual PGCs and all their daughter cells were tracked and observed.
Example 1
The embodiment discloses a method for constructing a stilla-CreERT 2 animal model.
In this example, the mouse construction strategy is shown in FIG. 1, in which the P2A-CreERT2 sequence is knocked in at a site before the stop codon of the stilla gene to form an open reading frame encoding the stilla-CreERT 2 fusion peptide. After translation is completed, the P2A polypeptide "self-cleaves" to produce STELLA protein and Creet 2 fusion protein, avoiding the effect on endogenous STELLA protein function. The CreERT2 fusion protein contains a mutant of the ligand binding region of the Cre recombinase and the estrogen receptor. In the absence of tamoxifen, the CreERT2 fusion protein localizes to the cytoplasm and has no Cre enzyme activity. After tamoxifen is given, the metabolite 4-OHT can be combined with Creet 2 to promote protein to enter the nucleus to perform Cre enzyme activity.
The specific method comprises the following steps:
1. Obtaining the target Gene
Name of the gene of interest (Ensembl number): stella (ENSMUSG 00000046323)
Target gene Ensembl website link ：http://asia.ensembl.org/Mus_musculus/Gene/Summarydb＝core;g＝ENSMUSG00000046323;r＝6:122603369-122607231;t＝ENSMUST00000049644
Transcripts for which the protocol was directed (Ensembl number): stella-201 (ENSMUST 00000049644.9)
2. Design of sgrnas:
the sgRNA was designed via the crispor.tefor.net website according to the sequence of the stilla gene as shown in SEQ ID No. 4.
SEQ ID NO.4：GCTTACATTGTACGCTGCCCTGG；
3. Preparation of sgrnas and Cas9 mRNA
The sgRNA and Cas9 mRNA of the Stella gene are prepared by in vitro transcription, and are sub-packaged in a refrigerator at-80 ℃ for standby.
4. Constructing a homologous recombination vector. A homologous recombination vector was constructed by the method of In-Fusion cloning, and the vector contained a 3.0kb 5 'homology arm, a P2A-Creert2 fragment, and a 3.0kb 3' homology arm. The sequences of the 5 'homology arm, the P2A-CreERT2 fragment and the 3' homology arm are respectively shown in SEQ ID NO. 1-3. The vector construction strategy is shown in FIG. 2, and the above 3 fragments are fused and then ligated with vector pBR322 through information. The primer sequences are shown in Table 1. The HindIII digestion identification recombination results are shown in FIG. 3, and the theoretical band sizes are 7359bp, 3825bp and 1233bp.
TABLE 1 primer sequences
5. In vitro injection of fertilized eggs and embryo transfer.
Preparing fertilized eggs of C57BL/6J mice, microinjecting Cas9 mRNA, sgRNA and homologous recombinant plasmids into the fertilized eggs, and transplanting the fertilized eggs into a C57BL/6J pregnant female mouse body to obtain F0 generation mice. Wherein, cas9 mRNA, sgRNA and homologous recombination plasmid concentrations are shown in table 2, fertilized eggs were obtained after 0.5 days of hybridization in C57BL/6J mice.
TABLE 2 microinjection liquid composition and concentration
6. Genotyping of F0 mice
(1) Primer design and identification scheme
To identify the genotype of the F0 mice, 2 pairs of primers (I and II, III and IV) were designed, the primer sequences are shown in Table 3, the identification strategy is shown in FIG. 4, and the identification scheme is shown in Table 4.
TABLE 3 primer sequences for genotyping mice
Table 4 mouse genotyping protocol
(2) Authentication method
PCR was performed using TAKARA PRIMESTAR GXL DNA Polymerase (R050A) to prepare a PCR reaction system according to Table 5, and a PCR reaction program was set according to Table 6. The PCR products were then subjected to agarose gel electrophoresis, and the results are shown in FIG. 5. The 5' arm (primers I and II) homologous recombination positive genomes should amplify a 5.2kb fragment and the negative genomes should amplify a 6.7kb fragment; the 3' arm (primers III and IV) homologous recombination positive genomes should amplify a 3.5kb fragment and the negative genomes should amplify a 7.0kb fragment. FIG. 5 shows that mouse No. 14 is a P2A-CreERT2 knock-in positive mouse.
TABLE 5PCR reaction System
TABLE 6PCR reaction procedure
7. F1 generation mice acquisition and genotyping
Because the early cleavage speed of fertilized eggs is very fast, the obtained F0 generation mice are chimeric and do not have the capability of stable inheritance, and the F1 generation mice which can be inherited stably need to be passaged.
Mating the F0 generation positive mice with wild C57BL/6J mice, breeding to obtain F1 generation mice, and carrying out genotype identification by PCR identification and sequencing. The PCR identification strategy and method are the same as those of the F0 generation mouse identification part, and the result of the PCR identification electrophoresis of the F1 generation mouse is shown in figure 6. The 5' arm (primers I and II) homologous recombination positive genomes should amplify a 5.2kb fragment and the negative genomes should amplify a 6.7kb fragment; the 3' arm (primers III and IV) homologous recombination positive genomes should amplify a 3.5kb fragment and the negative genomes should amplify a 7.0kb fragment. FIG. 6 shows that mice Nos. 3, 4, 6, 7, 11, 12, and 14 are P2A-CreERT2 knock-in positive mice.
To further confirm the correctness of the knockin sequence, the F1 generation positive mouse PCR products were sequenced and a total of 4 sequencing reactions were performed. Wherein, 5' homology arms are identified, and 2 sequencing reactions are performed on the sequencing of PCR products, which are respectively marked as follows: 1. 2;3' homology arm identification, 2 sequencing reactions were performed on the PCR product sequencing, and the sequencing reactions were respectively marked as follows: 3. 4. The sequencing results are shown in FIGS. 7-10 (Query is the target sequence, subject is the sequencing result)
Based on genotyping and sequencing results, 7P 2A-CreERT2 knock-in F1 mice were obtained, numbered 3,4, 6, 7, 11, 12, 14, respectively.
8. Subsequent offspring mouse genotyping
In the subsequent mating propagation process of mice, the genotypes of the mice can be identified by a short-fragment PCR method. The sequences of the primers for genotyping the mice are shown in Table 7.
TABLE 7 primer sequences for genotyping mice
A PCR reaction system was prepared using TAKARA TAQ DNA Polymerase (R001A) according to Table 8, followed by PCR reactions according to the PCR reaction program set forth in Table 9.
TABLE 8PCR reaction System
TABLE 9PCR reaction procedure
The PCR products were subjected to agarose gel electrophoresis, and the results are shown in FIG. 11.
Genotype judgment: after the genome of the mouse is amplified by using the primer pair (P1, P2) and (P3, P4), the sizes of products are 463bp and 645bp respectively, and the wild mouse only amplifies 463bp bands; heterozygote mice can amplify 463bp and 645bp bands; homozygous mice can only amplify 645bp bands.
Embodiment two:
this example provides an efficiency test of the villa-CreERT 2 mouse Cre enzyme.
Mouse STELLA was specifically expressed in PGCs from day E7.25, up to day E13.5 in female embryonic germ cells, and up to day E16.5 in male embryonic germ cells. Rosa26-tdTomato report mice, which are a common model for verifying Cre expression efficiency, were constructed by inserting CAG-LSL (loxp-3×stop-loxp) -tdTomato sequences into the mouse Rosa26 gene locus. In the absence of Cre enzyme, mice were unable to express red fluorescent protein due to the presence of stop cassette LSL between CAG promoter and TdTomato sequences; in the presence of Cre enzyme, cre enzyme cleaves the 3 XStop sequence between the two loxp sites, thereby allowing TdTomato to express and emit red fluorescence (PMID: 20023653). Therefore, we tested the efficiency of the stilla-CreERT 2 mouse Cre enzyme by means of Rosa 26-tdmamato report mice.
Adult Stella-Creet 2 mice of F1 generation were caged with Rosa26-tdTomato mice, and the next day the bolts were checked, and bolts were seen at 12 pm as pregnant 0.5 (E0.5) days. At day E7.5 pregnant mice were intraperitoneally injected with tamoxifen (0.075 mg/g) in a single injection to drive Cre enzyme into the nucleus for action. Pregnant mice were sacrificed at day E12.5, embryo genotypes were collected and identified, frozen sections of the STELLA protein immunofluorescent staining and TdTomato fluorescent protein detection were performed on the control (R26 tdT/+) and embryo of interest (R26 tdT/+; stella-Creet 2) genital ridges, and TdTomato expression profiles and co-localization with STELLA were observed. The results are shown in FIG. 12, no TdTomato expression in STELLA green fluorescence positive PGCs in R26tdT/+ embryos; r26tdT/+; the specific expression of TdTomato in the genital ridge is visible in the stilla-CreERT 2 embryo, no positive signal exists in other tissues, and the co-standard rate of the stilla green fluorescence can reach 83.96%, which indicates that the stilla-CreERT 2 mouse Cre enzyme is specifically expressed in PGC, other tissues have no obvious leakage, and the stilla-CreERT 2 mouse Cre enzyme can be used for the construction of a mouse PGC inducible gene knockout mouse model in the follow-up.
Embodiment III:
This example provides the use of a stilla-CreERT 2 mouse for the spatiotemporal specific knockout of the Mcm8 gene in inducible PGCs.
MCM8 is a member of the Mini Chromosome Maintenance (MCM) protein family, playing an important role in maintenance of genomic stability. At present, genetic research shows that MCM8 is closely related to human ovary function, animal experiments also show that MCM8 knockout leads to increased DNA damage in the meiosis process of mice, homologous chromosome association is abnormal, and female mice are sterile. We and other subject groups found multiple MCM8 gene mutations in premature ovarian dysfunction (Premature ovarian insufficiency, POI) families and sporadic patients, and functional experiments demonstrated that these mutations resulted in abnormal DNA damage repair capability. In summary, it is currently believed that mutations in this gene cause abnormalities in homologous recombination repair during meiosis leading to the occurrence of POIs. But MCM8 plays an important role in maintenance of mitotic genomic stability as well, and it is highly expressed in human PGCs prior to meiosis, suggesting that MCM8 may play an important role in PGC development in addition to meiosis. We therefore used the stilla-CreERT 2 mice to make specific and inducible knockouts of the Mcm8 gene in PGCs to clarify its role in PGC development. Adult stilla-CreERT 2, mcm8-flox mice were caged, and the next day were checked for embolism, and those who were found to be embolism at 12 noon were treated as E0.5 days. At day E7.5, single injection of tamoxifen (0.075 mg/g) into the abdominal cavity of pregnant mice induced Mcm8 gene knockout. Pregnant mice were sacrificed at day E12.5, embryos were obtained and genotypes were identified, and as shown in FIG. 13, the mouse genome was amplified using the Mcm8-flox primer pair, wild-type mice amplified only 282bp bands, mcm8-flox heterozygote mice amplified 282bp and 348bp bands, and Mcm8-flox homozygote mice amplified only 348bp bands. The mouse genome was amplified using the stilla-CreERT 2 primer pair P3, P4, the stilla-CreERT 2 positive genome should amplify a 645kb band, and the negative genome cannot amplify a band. Mcm8 +/+; the Stella-CreERT2 is a Mcm8-flox wild type, stella-CreERT2 positive embryo; mcm8 fl/+; the Stella-CreERT2 is Mcm8-flox heterozygous, stella-CreERT2 positive embryo; mcm8 fl/fl is a Mcm8-flox homozygous, stella-CreERT2 negative embryo; Mcm8 fl/fl; the stilla-CreERT 2 is a Mcm8-flox homozygous stilla-CreERT 2 positive embryo. Alkaline phosphatase staining was then performed to observe the number of PGCs for embryos of different genotypes, as shown in fig. 14, mcm8 fl/fl; the number of PGCs in the stilla-CreERT 2 embryo is obviously reduced compared with that of the Mcm8 fl/fl embryo, which shows that the specific knockout of MCM8 in the PGCs can influence the development of the PGCs, and further, the model can be used for researching the development regulation mechanism of the PGCs.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and are not limiting. Although the present invention has been described in detail with reference to the given embodiments, those skilled in the art can make modifications and equivalents to the technical solutions of the present invention as required without departing from the spirit and scope of the technical solutions of the present invention.

Claims (8)

1. The method for constructing the stilla-CreERT 2 animal model is characterized by comprising the following steps of:
1) Constructing homologous recombinant plasmids;
2) Injecting Cas9 mRNA, sgRNA and homologous recombinant plasmid into fertilized eggs of animals through microinjection, and transplanting the fertilized eggs after injection into a gestation animal body to obtain F0-generation animals;
3) Mating the F0 generation animal with a wild type animal to obtain an F1 generation;
4) The F1 generation is identified as positive and is a Stella-CreERT2 animal;
wherein the model animal is a mouse;
the homologous recombinant plasmid comprises a 5 'homology arm, a P2A-CreERT2 fragment and a 3' homology arm which are connected in sequence;
The nucleic acid sequences of the 5 'homology arm, the P2A-CreERT2 fragment and the 3' homology arm are respectively shown in SEQ ID NO. 1-3;
The target of the sgRNA is located on the fourth exon of the stilla gene; ensembl number ENSMUST00000049644.9 of the stilla gene;
the sgRNA nucleic acid sequence is shown as SEQ ID NO. 4.
2. The method of constructing a stilla-CreERT 2 animal model according to claim 1, wherein the animal fertilized egg is a C57BL/6J mouse fertilized egg;
the fertilized eggs of the C57BL/6J mice are obtained after hybridization of male and female C57BL/6J mice and fertilization for 0.5 day.
3. Use of the method of constructing a stilla-CreERT 2 animal model according to claim 1 or 2 in any one of the following:
1) Knocking out genes related to embryo PGC development;
2) For modulating PGC development;
3) For constructing PGC dysplasia animal models;
4) Used for regulating embryo development;
5) For constructing an animal model of embryo dysplasia;
6) Primordial germ cell PGC lineage tracing;
7) And (3) researching the action and mechanism of PGC development related genes.
4. The use according to claim 3, wherein the PGC development-related gene is the minichromosome maintenance encoding gene 8.
5. A construction method of a Mcm8 gene knockout model mouse is characterized in that a stilla-CreERT 2 animal according to claim 1 is hybridized with a Mcm8-flox mouse, and the Mcm8 gene knockout model mouse Mcm8 fl/fl in PGC is obtained by injecting tamoxifen into the abdominal cavity of the pregnant mouse to induce Mcm8 gene knockout.
6. The method of claim 5, wherein the tamoxifen is induced at a concentration of 0.075 mg/g/min.
7. Use of the method for constructing Mcm8 knockout model mice according to claim 5 or claim 6 in any one or more of the following:
i) For modulating PGC development;
II) is used for constructing a PGC dysplasia mouse model;
III) for modulating embryo development;
IV) for constructing an embryonic dysplasia mouse model;
v) PGC lineage tracing;
VI) research on the action and mechanism of PGC development related genes.
8. A kit for constructing a stilla-CreERT 2 animal model based on CRISPR-Cas9 technology, characterized in that the kit comprises Cas9 mRNA, sgRNA and homologous recombinant plasmid of claim 1;
wherein the Cas9 mRNA is obtained by in vitro transcription of a Cas9 nuclease.
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