CN116790662A - Construction method and application of sheep VASA gene report vector - Google Patents
Construction method and application of sheep VASA gene report vector Download PDFInfo
- Publication number
- CN116790662A CN116790662A CN202310501774.1A CN202310501774A CN116790662A CN 116790662 A CN116790662 A CN 116790662A CN 202310501774 A CN202310501774 A CN 202310501774A CN 116790662 A CN116790662 A CN 116790662A
- Authority
- CN
- China
- Prior art keywords
- vasa
- vector
- primer
- plasmid
- sgrna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 101150089602 vasa gene Proteins 0.000 title claims abstract description 87
- 239000013598 vector Substances 0.000 title claims abstract description 71
- 241001494479 Pecora Species 0.000 title claims abstract description 24
- 238000010276 construction Methods 0.000 title claims abstract description 21
- 108091033409 CRISPR Proteins 0.000 claims abstract description 51
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 39
- 108020005004 Guide RNA Proteins 0.000 claims abstract description 30
- 210000004027 cell Anatomy 0.000 claims abstract description 24
- 230000014509 gene expression Effects 0.000 claims abstract description 20
- 230000004913 activation Effects 0.000 claims abstract description 11
- 239000013612 plasmid Substances 0.000 claims description 56
- 239000012634 fragment Substances 0.000 claims description 29
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 17
- 108020004414 DNA Proteins 0.000 claims description 16
- 210000004602 germ cell Anatomy 0.000 claims description 15
- 239000013613 expression plasmid Substances 0.000 claims description 14
- 238000001976 enzyme digestion Methods 0.000 claims description 12
- 238000005520 cutting process Methods 0.000 claims description 11
- 238000003198 gene knock in Methods 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 10
- 230000003213 activating effect Effects 0.000 claims description 9
- 239000012190 activator Substances 0.000 claims description 8
- 230000002194 synthesizing effect Effects 0.000 claims description 8
- 230000008685 targeting Effects 0.000 claims description 7
- 230000011712 cell development Effects 0.000 claims description 5
- 241000894007 species Species 0.000 claims description 5
- 238000000137 annealing Methods 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 238000007877 drug screening Methods 0.000 claims description 3
- 230000008844 regulatory mechanism Effects 0.000 claims 1
- 239000000523 sample Substances 0.000 claims 1
- 210000002950 fibroblast Anatomy 0.000 abstract description 16
- 101000830411 Homo sapiens Probable ATP-dependent RNA helicase DDX4 Proteins 0.000 abstract description 12
- 108090000623 proteins and genes Proteins 0.000 abstract description 12
- 238000000338 in vitro Methods 0.000 abstract description 10
- 238000011160 research Methods 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 7
- 238000001890 transfection Methods 0.000 abstract description 7
- 230000006870 function Effects 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 description 9
- 102100024770 Probable ATP-dependent RNA helicase DDX4 Human genes 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000011161 development Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 230000003321 amplification Effects 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 101150063416 add gene Proteins 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 3
- 210000002149 gonad Anatomy 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 210000001550 testis Anatomy 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000003147 molecular marker Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 238000003822 preparative gas chromatography Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000021595 spermatogenesis Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- -1 22208) as a template Proteins 0.000 description 1
- 101150039504 6 gene Proteins 0.000 description 1
- 101150087690 ACTB gene Proteins 0.000 description 1
- 241000238097 Callinectes sapidus Species 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001037191 Homo sapiens Hyaluronan synthase 1 Proteins 0.000 description 1
- 102100040203 Hyaluronan synthase 1 Human genes 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 208000007466 Male Infertility Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001280406 Mauremys mutica Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 101710136296 Probable ATP-dependent RNA helicase DDX4 Proteins 0.000 description 1
- 241000242678 Schistosoma Species 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 230000011559 double-strand break repair via nonhomologous end joining Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000004420 female germ cell Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 101150044508 key gene Proteins 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 210000003794 male germ cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004879 molecular function Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000006780 non-homologous end joining Effects 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000000920 spermatogeneic effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
Abstract
The invention discloses a construction method and application of a sheep VASA gene report vector, and aims to construct the VASA gene report vector by using a CRISPR/Cas9 technology and verify the effectiveness of a knock-in vector in fibroblasts by using a CRISP/dCAS9 system. The construction method of the vector of the invention comprises the following steps: (1) constructing an EGFP-PGK puro-VASA report vector; (2) designing a target gRNA sequence based on a VASA gene exon sequence, constructing a Cas9-gRNA knock-in vector, and carrying out fibroblast transfection together; (3) the CRISPR/dCas9 activation system was used to activate VASA gene expression. The vector of the invention can be used for verifying the effectiveness in vitro cells. The invention can realize the activation expression of the exogenous gene VASA in sheep fibroblasts, and provides a research basis for further exploring the functions of reproduction specific genes.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a construction method and application of a sheep VASA gene report vector.
Background
The VASA gene is a member of the DEAD-box family of genes encoding ATP-dependent RNA helicases, also known as DDX4 or MVH, originally identified in drosophila, and studies have shown that the VASA gene is highly conserved in invertebrates and vertebrates, and that the molecular functions of the VASA protein include: can bind to target mRNA associated with germ cell establishment and control translation initiation during egg production. Currently, the VASA gene has been widely studied in germ cells of drosophila, fish, crabs, mammals, etc. In research on the origin and migration of fish primordial germ cells (Primordial germ cells, PGCs), the VASA gene can be used as a marker to trace the whole development process of PGCs, and is expressed in the whole spermatogenesis process. The VASA gene is expressed in both ovary and testis of blue crab, and the VASA expression shows dynamic change in spermatogenesis process and is mainly expressed in spermatogonia and primary spermatocyte. In humans, VASA is expressed in the cytoplasmic membranes of the head and tail of sperm and significantly reduced in oligospermic men, and thus, VASA can be a molecular marker for male infertility. The VASA homolog (MVH) gene homozygous mutant mice show reproductive defects in a sex-dependent manner, and the MVH homozygous mutation can lead to spermatogenic defects of male mice, cause meiosis injury, apoptosis and the like in testes, lead to abnormal development of male gonads in embryos, and do not influence the development of female germ cells. The VASA gene is expressed in human fetus and adult gonad, and is mainly expressed in spermatocyte and mature oocyte, and is the high specificity marker gene of human germ cell. Furthermore, the expression of the VASA gene is also different in different species, for example higher in female schistosome than in male; in the different embryos of the Asian yellow pond turtle, the expression level in the ovary is higher than that of the testis, but the opposite is true after birth. Thus, the VASA gene is considered as a molecular marker for identification of various animal germ cell lineages and is a key gene for research of germ cell differentiation and development.
The CRISPR/Cas9 system comprises Cas9 protein and guide RNA (gRNA), wherein the Cas9 protein plays a role in DNA double-strand cutting, the gRNA plays a role in guide recognition, and DNA double-strand breakage (DNAdouble strand breaks, DSB) is followed by self-repair by utilizing homology directed repair (homology directed repair, HDR), micro-homology-mediated end connection (micro-mediated end joining, MMEJ) or non-homology end connection (non-homologous end joining, NHEJ) so as to complete targeted mutation, insertion, deletion or gene knockout of genes and realize modification of genome. The CRISPR/Cas9 gene editing technology has the advantages of high targeting efficiency, low off-target effect, low technical difficulty and the like, and is widely used for researches such as disease control, livestock breeding and the like. The CRISPR/Cas9 knocking-in system can effectively knock in exogenous DNA into target genome sites, promote the function exertion of target genes and achieve the aim of editing genome. Currently, CRISPR/Cas9 gene knock-in technology based on has successfully constructed knock-in cell lines and mouse models for multiple genes.
The VASA gene is a germ cell marker, and research on its effect on germ cell development is crucial, but little research is done on sheep VASA gene. The invention constructs pEGFP-PGK puro-VASA knock-in plasmid based on CRISPR/Cas9 system, further verifies knock-in efficiency by using CRISPR/dCAs9 technology, successfully constructs a sheep VASA gene report vector, and lays a foundation for researching the action mechanism of VASA gene in sheep germ cell in-vitro induced differentiation and development.
Disclosure of Invention
The VASA gene is a germ line marker for research of germ cell and gonad origin and development, and there is currently less exploration of the VASA gene in sheep. The invention aims to construct a VASA gene report vector by using a CRISPR/Cas9 system, and adopts a CRISPR/dCAS9 technology to activate the VASA gene, so that the VASA gene can be successfully activated and expressed in sheep fibroblasts, and a reference basis is provided for further exploring a regulation and control mechanism of the VASA gene in sheep germ cell development in the future.
In order to achieve the above object, the present invention provides the following technical solutions:
a VASA gene reporter vector comprising a recombinant EGFP-PGK puro-VASA plasmid, a Cas9-gRNA plasmid, and a CRISPR dCas9 activation vector;
the recombinant EGFP-PGK puro-VASA plasmid is obtained by adopting the following method: according to sequence information near 24 th exons of VASA genes, a PAM locus is found, 5 'and 3' homology arm primers near the VASA gene knock-in locus are respectively designed, genome DNA of a target species (such as sheep) is used as a template, homologous arm fragments containing enzyme cutting loci are obtained through amplification, and the homologous arm fragments are connected to a pMD-19T vector to obtain a pMD-5&3HA intermediate vector; designing primers by taking TV-hPITX3-Reverse (Addgene, 22208) as a template, amplifying by PCR to obtain EGFP-LoxP-PGK-Puro-LoxP fragments, and connecting the EGFP-LoxP-PGK-Puro-LoxP fragments with a linearized pMD-5&3HA intermediate vector to obtain recombinant EGFP-PGK Puro-VASA plasmids;
the Cas9-gRNA plasmid is obtained by adopting the following method: according to the PAM locus, a gRNA primer is designed and synthesized, the synthesized gRNA primer is annealed and subjected to enzyme digestion, and then linearized pX330 plasmid (Addgene, 42230) is inserted in a connection mode, so that a Cas9-gRNA plasmid is obtained;
the CRISPR dCas9 activation vector is obtained by the following method: designing and synthesizing sgRNA primers targeting the VASA gene promoter; the sgRNA primer is annealed and is subjected to enzyme digestion, and then is connected and inserted into a linearized dCAS9-p300 plasmid (Addgene, 61361), so that the CRISPR/dCAS9-p300 Activator expression plasmid is successfully constructed.
As a preferred embodiment, the nucleotide sequences of the 5 'and 3' homology arm primers near the VASA gene knock-in site are as follows:
5’homologous arm-F：GCTAGCATCCCATGACTCATCATCTA(SEQ ID NO.1)
5’homologous arm-R：TCATCCAGCCCAGCATTTTG(SEQ ID NO.2)
3’homologous arm-F：GGTACCCCCCTGTATTTATGTCTCACTTGTT(SEQ ID NO.3)
3’homologous arm-R：CTCGAGGATGCAGAGAAGAAAGTAG(SEQ ID NO.4)；
the nucleotide sequence of the EGFP-LoxP-PGK-Puro-LoxP fragment related primer is as follows:
P2A-EGFP-F：
CAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTGTGAGCAAGGG CGAGGAGCT(SEQ ID NO.5)
XHOI-LOXP-PURO-R：
ACTCTCGAGATAACTTCGTATAATGTATGCTATACGAAGTTATGAACCTCTTCGAGGGAC CTA(SEQ ID NO.6)
NHEI-P2A-F：
TCAGCTAGCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCA(SEQ ID NO.7)。
as a preferred embodiment, the gRNA primer is a gRNA3 primer or a gRNA4 primer, and the nucleotide sequences of the upstream primer and the downstream primer of the gRNA3 primer and the gRNA4 primer are as follows:
VASA-gRNA3-F：tttcttggctttatatatcttgtggaaaggacGAAAACAACCTGCAAGTTTG(SEQ ID NO.8)
VASA-gRNA3-R：tcaacttgctatgctgtttccagcatagctctgaaacCAAACTTGCAGGTTGTTTTC(SEQ ID NO.9)
VASA-gRNA4-F：tttcttggctttatatatcttgtggaaaggacgaaacaccGACTCATCATCTACTGGATT(SEQ ID NO.10)
VASA-gRNA4-R：tcaacttgctatgctgtttccagcatagctctgaaacAATCCAGTAGATGATGAGTC(SEQ ID NO.11)。
as a preferred technical scheme, the sgRNA primer is at least one of the sgRNA-a and the sgRNA-b, and the nucleotide sequences of the sgRNA-a and the sgRNA-b are as follows:
sgRNA-a：TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAACTGTACC ACAAACTTGC(SEQ ID NO.12)
sgRNA-b：TCAACTTGCTATGCTGTTTCCAGCATAGCTCTGAAACGCAAGTTTGTGGT ACAGTTC(SEQ ID NO.13)。
the construction method of the VASA gene report vector also belongs to the protection scope of the invention, and the VASA gene report vector comprises a recombinant EGFP-PGK puro-VASA plasmid, a Cas9-gRNA plasmid and a CRISPR dCas9 activation vector;
(1) The construction method of the recombinant EGFP-PGK puro-VASA plasmid comprises the following steps: designing 5 'and 3' homology arm primers near a VASA gene knock-in site respectively, and amplifying by taking target species genome DNA as a template to obtain homology arm fragments containing enzyme cutting sites, and connecting the homology arm fragments to a pMD-19T vector to obtain a pMD-5&3HA intermediate vector; designing primers by taking TV-hPITX3-Reverse as a template, amplifying by PCR to obtain EGFP-LoxP-PGK-Puro-LoxP fragments, and connecting the EGFP-LoxP fragments with a linearized pMD-5&3HA intermediate vector to obtain recombinant EGFP-PGK Puro-VASA plasmids;
(2) The construction method of the Cas9-gRNA plasmid comprises the following steps: according to the PAM locus, designing and synthesizing a gRNA primer, annealing the synthesized gRNA primer, performing enzyme digestion, and connecting and inserting the synthesized gRNA primer into a linearized pX330 plasmid to obtain a Cas9-gRNA plasmid;
(3) The construction method of the CRISPR dCas9 activation vector comprises the following steps: designing and synthesizing sgRNA primers targeting the VASA gene promoter; the sgRNA primer is annealed and is subjected to enzyme digestion, and then is connected and inserted into the linearized dCAS9-p300 plasmid, so that the CRISPR/dCAS9-p300 Activator expression plasmid is successfully constructed.
The primers involved in the above construction method are as described above.
The application of the VASA gene report vector in knocking-in and activating expression of the VASA gene also belongs to the protection scope of the invention.
The application of the VASA gene report vector in researching the regulation and control mechanism of the VASA gene involved in sheep germ cell development also belongs to the protection scope of the invention.
A method for reporting and activating expression of a VASA gene, wherein the VASA gene reporting vector is used for knocking in and activating the VASA gene. As a preferred technical scheme, a target tool cell is transfected with a recombinant pEGFP-PGK puro-VASA plasmid and a Cas9-gRNA plasmid together, puro medicine screening is carried out, then the CRISPR dCas9 activating vector is adopted to transfect the screened cell, and the expression of a VASA gene in the cell is activated.
The invention constructs a knock-in vector of a VASA gene by using CRISPR/Cas9 technology, and verifies the effectiveness of the knock-in vector in fibroblasts by using a CRISPR/dCAS9 system. The construction method of the vector of the invention comprises the following steps: (1) constructing an EGFP-PGK puro-VASA vector and carrying out fibroblast transfection; (2) designing a target gRNA sequence based on a VASA gene exon sequence, and constructing a Cas9-gRNA knock-in vector; (3) the CRISPR/dCas9 activation system was used to activate VASA gene expression.
The vector of the invention can be used for verifying the effectiveness in vitro cells. The invention can realize the activation expression and tracing of VASA-EGFP synthetic genes in sheep fibroblasts, and provides a research basis for further exploring the functions of reproduction specific genes.
The invention has the beneficial effects that:
the invention utilizes CRISPR/Cas9 technology to construct VASA gene report carrier in vitro, successfully transfers into sheep fibroblasts, and further combines CRISPR/dCAS9 system to activate the expression of VASA gene, thereby proving the effectiveness of the VASA gene report carrier and providing reference for the next research of the development and differentiation of sheep male germ cells by VASA gene.
Drawings
Fig. 1: the pEGFP-PGK puro-VASA vector is constructed schematically and structurally.
Fig. 2: gel diagram of amplification products of homologous arms on two sides of pEGFP-PGK puro-VASA vector.
Fig. 3: VASA gene gRNA targeting site and in vitro guided cutting verification gel diagram.
Fig. 4: the VASA gene Cas9-gRNA vector construction schematic diagram and gRNA sequence insertion sequencing identification.
Fig. 5: gel map of amplified product after in vitro transfection of the VASA gene reporter vector by fibroblasts.
Fig. 6: sgRNA design of targeted VASA genes.
Fig. 7: the fibroblast cells were transfected with VASA gene sgRNA vector in vitro to activate VASA gene expression product gel pattern and fluorescence expression pattern.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent suppliers. The primers related to the embodiment of the invention are synthesized by Nanjing qingke biotechnology Co.
Example 1: construction and verification of fluorescence report carrier EGFP-PGK puro-VASA
According to the VASA gene sequence information in NCBI database, two PAM sites are determined near the 24 # exon, 5 'and 3' homology arm primers near the VASA gene knock-in site are respectively designed, sheep genome DNA is used as a template, homologous arm fragments containing enzyme cutting sites are obtained through amplification, and the homologous arm fragments are connected to a pMD-19T vector to obtain a pMD-5&3HA intermediate vector; designing primers by taking TV-hPITX3-Reverse (Addgene, 22208) as a template, amplifying by PCR to obtain EGFP-LoxP-PGK-Puro-LoxP fragments, and connecting the EGFP-LoxP-PGK-Puro-LoxP fragments with a linearized pMD-5&3HA intermediate vector to obtain recombinant EGFP-PGK Puro-VASA plasmids;
the nucleotide sequences of the 5 'and 3' homology arm primers near the VASA gene knock-in site are shown in table 1:
TABLE 15 'and 3' homology arm primer sequences near the VASA gene knock-in site
The nucleotide sequence of EGFP-LoxP-PGK-Puro-LoxP fragment-related primer is as follows:
P2A-EGFP-F：CAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGAC CTGTGAGCAAGGGCGAGGAGCT
XHOI-LOXP-PURO-R：ACTCTCGAGATAACTTCGTATAATGTATGCTATACGAAGTT ATGAACCTCTTCGAGGGACCTA
NHEI-P2A-F：TCAGCTAGCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCA。
the detailed procedure is as follows:
1. the sheep genome DNA is used as a template, 5'homologous arm-F and 5' homologous arm-R are used as primers for amplification to obtain a 5 'Homology Arm (HA) fragment containing an enzyme cutting site, the 5' Homology Arm (HA) is connected to pMD-19T, and then an M13-47F primer (F: CGCCAGGGTTTTCCCAGTCACGAC, R: CACACAGGAAACAGCTATGAC) is selected for sequencing to extract plasmids from bacterial liquid in a forward direction.
2. Sheep genome DNA is used as a template, 3'homologo arm-F and 3' homologo arm-R are used as primers for amplification to obtain a 5 'Homology Arm (HA) fragment containing an enzyme cutting site, after 3' HA is connected to pMD-19T, sequencing is carried out at two ends, and plasmids containing KpnI bacterial solutions at two ends are extracted.
3. The linearized pMD-5HA and 3' HA were recovered by cleavage of pMD-3HA and pMD-5HA using KpnI, then ligated using T4-ligase, transformed, and sequenced to give the correct orientation of pMD-5&3HA.
4. Using TV-hPITX3-Reverse (Addgene, 22208) as a template, P2A (post) -EGFP-LoxP-PGK-PURO-LoxP was amplified using the P2A-EGFP-F and XHOI-LOXP-PURO-R primers with high fidelity enzyme reference, and then the amplified product was taken out to dilute ten times directly, and P2A-EGFP-LoxP-PGK-PURO-LoxP was amplified using 1. Mu.L as a template using NheI-P2A-F and XhoI-LOXP-PURO-R primers, followed by PCR product recovery.
5. Then, the NheI and XhoI are used for double digestion of pMD-5&3HA and P2A-EGFP-LoxP-PGK-Puro-LoxP PCR to recover products, then the digested products are recovered and then connected by T4 ligase to obtain the target product pVASA-EGFP-LoxP-PGK-Puro-LoxP (i.e. recombinant EGFP-PGK Puro-VASA plasmid).
The construction schematic diagram and the vector map of the recombinant pEGFP-PGK puro-VASA vector are shown in figure 1; the vector was subjected to gel electrophoresis, as shown in FIG. 2, indicating that the two homology arms were successfully amplified and met the target size.
Example 2: VASA gene gRNA design and construction of Cas9-gRNA expression plasmid
According to the PAM site information in the vicinity of exon 24 of the above VASA gene, the gRNA sequence was involved. This example is directed to design 3 grnas for exon 24 of the VASA gene, where gRNA3 is on the sense strand, grnas 1 and 4 are on the negative strand, the targeting sites for the three grnas are shown in fig. 3, and the sequence of each gRNA is shown in table 2.
TABLE 2VASA Gene gRNA sequences
Further adopting Cas9 and gRNA co-expression vector pX330 constructed in Zhang Feng laboratory, adding Bbs I viscous terminal at two ends of gRNA sequence, and synthesizing by Nanjing qingke biotechnology Co-Ltd. The synthesized gRNA primer is annealed, namely, the gRNA is treated by T4 PNK, and PCR reaction is carried out, wherein the temperature is 37 ℃ for 30min, 95 ℃ for 5min, and the sample is preserved at the speed of 5 ℃/min from 95 ℃ to 25 ℃. The annealed samples were subjected to cleavage and ligation insertion into the pX330 plasmid (plasmid construction reaction system and cleavage ligation reaction system are shown in tables 3 and 4), i.e., 5min at 37℃and 5min at 21℃for 6 cycles. Finally, the enzyme digestion connection reaction product and Dh5α competent cells are mixed for plasmid transformation and sequencing, DNA is extracted, in vitro guided cleavage experiments are carried out, electrophoresis results prove that three gRNA bands are obvious, and sequencing shows that the guide sequences of three plasmids Cas9-g1, cas9-g3 and Cas9-g4 are inserted correctly (figure 4).
TABLE 3gRNA PX330 plasmid construction reaction System
Table 4 enzyme digestion ligation reaction system
Example 3: in vitro transfected VASA gene Cas9-gRNA and EGFP-PGK puro-VASA plasmid vector and identification
Collecting sheep ear tissue, sterilizing with 75% alcohol, cutting wool, separating epithelium tissue, cleaning for several times, and cutting into pieces of 1mm 2 Tissue adherence of small tissue pieces for fibroblast separation, and culture with DMEM medium containing 10% fetal calf serum and 1% diabody at 37deg.C and 5% CO 2 The incubator was incubated, and after approximately one week of incubation, the cells were successfully isolated and used for the next experiment.
To verify the effectiveness of the constructed knock-in vector, cas9-gRNA and EGFP-PGK puro-VASA plasmid transfection was performed on sheep ear fibroblasts,essentially comprising culturing cells in six-well plates at 37℃and 5% CO in DMEM medium containing 10% fetal bovine serum and 1% diabody 2 Culturing in incubator, and passaging until cell confluence reaches 95% or so and cell density reaches 70-80% or so3000 reagent (Invitrogen, L3000-008) shows that constructed Cas9-gRNA plasmid and pEGFP-PGK puro-VASA plasmid are transfected together, namely, the amount of plasmid DNA transfected by each six-well plate is 2.5 mug, drug screening is carried out after 48 hours of transfection, namely, cell culture is carried out by using culture solution containing antibiotic puro, liquid exchange is carried out every day, dead cells are removed, and the next experiment and DNA and RNA extraction are carried out after 3-5 days of cell growth. Collecting cell genome DNA for targeted cleavage experiment detection and amplification target band detection; as shown in fig. 5, T7E1 detection showed that Cas9-g3 and Cas9-g4 bands were evident, demonstrating that both grnas could successfully make genomic cleavage; PCR amplification results show that the fragment with 2627bp can be successfully amplified after transfection, and the VASA gene is proved to be successfully knocked into cells, and subsequent experiments utilize the Cas9-gRNA3 vector for transfection.
Example 4: CRISPR/dCAS9 activated VASA gene efficiency assay
To effectively activate the expression of the VASA gene in vitro, two sgRNAs (FIG. 6) for targeted activation of VASA were designed at the sgRNA design website based on the promoter sequence information of the VASA gene, and the sgRNA sequences are shown in the table.
TABLE 5VASA Gene sgRNA sequences
The BbsI is used for enzyme digestion of vector plasmid dCAS9-p300 at 37 ℃, agarose gel electrophoresis is carried out, a DNA fragment recovery kit is used for gel digestion and recovery of target vector fragments, a PCR instrument is used for primer annealing, enzyme ligation is carried out, plasmid conversion is carried out, plasmid extraction is carried out, and after sequencing by a company, target plasmid is extracted, thus successfully constructing CRISPR/dCAS9-p300 Activator expression plasmid. In the embodiment, the sgRNA-a and the sgRNA-b are respectively connected with a vector plasmid dCAS9-p300 after enzyme digestion, so as to respectively construct a CRISPR/dCAS9-p300 Activator expression plasmid containing the sgRNA-a and a CRISPR/dCAS9-p300 Activator expression plasmid containing the sgRNA-b;
the constructed CRISPR/dCAS9-P300 actor expression plasmid is transfected into sheep ear fibroblasts obtained by a prodrug screen, total RNA of the cells is extracted after 48 hours for further quantitative expression identification and fluorescence identification of VASA genes, and the result shows that when the CRISPR/dCAS9-P300 actor expression plasmid containing sgRNA-a or sgRNA-b is transfected singly, the effect is poor, simultaneously, the CRISPR/dCAS9-P300 actor expression plasmid containing sgRNA-a and the CRISPR/dCAS9-P300 actor expression plasmid containing sgRNA-b can effectively improve the expression level (P < 0.05) of the VASA genes, and meanwhile, the cells can show target fluorescence after being transfected by two sgRNAs (figure 7), and the expression of the VASA genes in the sheep ear fibroblasts can be effectively activated by simultaneously transfecting the two CRISPR/dCAS9-P300 actor expression plasmids.
The detailed process of cell DNA and RNA extraction and fluorescence quantitative PCR is as follows: after ear fibroblast cells are transfected with cas9-gRNA and recombinant pEGFP-PGK puro-VASA plasmid for 48 hours, the cells are further screened by puro medicine, the culture solution is sucked and washed three times by DPBS after the cells are converged to about 95%, and the genomic DNA of the cells is extracted and purified by referring to reagent instructions, and then the next PCR experiment is carried out; after the drug screening, ear fibroblasts were transfected with CRISPR/dCAS9-p300 Activator expression plasmid for 48h, cells were lysed by adding Trizol reagent, transferred to a 1.5mL centrifuge tube after full lysis, RNA extracted according to the instructions, and cDNA synthesis according to the Norvezan reverse transcription instructions. VaSA gene primers were designed using NCBI database and synthesized by Nanjing qing Biotechnology Co. Fluorescent quantitative experiments were performed according to the Renzan SYBR qPCR fluorescent quantitative premix enzyme instructions, wherein the reaction system comprises: SYBR qPCR Master Mix 10. Mu.L, 0.6. Mu. L, cDNA 1. Mu.g each of the gene upstream and downstream primers (10. Mu. Mol. L-1), nuclease Free Water were supplemented to 20. Mu.L. The reaction system was reacted in a fluorescent quantitative PCR apparatus with reference to the following conditions: 50 ℃ for 2min; pre-denaturation at 95℃for 10min; sequentially carrying out 40 cycles at 95 ℃ for 15s, 60 ℃ for 30s and 72 ℃ for 30 s; finally, the extension is carried out for 10min at 72 ℃. Results dataThrough 2 -ΔΔCt In this method, the expression level of the VASA gene was analyzed by relative quantification using the ACTB gene as a reference gene (see Table 6 for sequences).
TABLE 6 Gene primer sequences
Data processing and analysis: the experiments according to the invention were repeated at least 3 times and the test data were expressed as mean.+ -. Standard Error (SEM). SPSS (24) software performs a difference significance analysis on the data, with x,/x and x representing difference significance P <0.05, P <0.01 and P <0.001, respectively.
Claims (10)
1. A VASA gene reporting vector, characterized in that the VASA gene reporting vector comprises a recombinant EGFP-PGKpuro-VASA plasmid, a Cas9-gRNA plasmid and a CRISPR dCas9 activating vector;
the recombinant EGFP-PGK puro-VASA plasmid is obtained by adopting the following method: designing 5 'and 3' homology arm primers near a VASA gene knock-in site respectively, and amplifying by taking target species genome DNA as a template to obtain homology arm fragments containing enzyme cutting sites, and connecting the homology arm fragments to a pMD-19T vector to obtain a pMD-5&3HA intermediate vector; designing primers by taking TV-hPITX3-Reverse as a template, amplifying by PCR to obtain EGFP-LoxP-PGK-Puro-LoxP fragments, and connecting the EGFP-LoxP fragments with a linearized pMD-5&3HA intermediate vector to obtain recombinant EGFP-PGK Puro-VASA plasmids;
the Cas9-gRNA plasmid is obtained by adopting the following method: according to the PAM locus, designing and synthesizing a gRNA primer, annealing the synthesized gRNA primer, performing enzyme digestion, and connecting and inserting the synthesized gRNA primer into a linearized pX330 plasmid to obtain a Cas9-gRNA plasmid;
the CRISPR dCas9 activation vector is obtained by the following method: designing and synthesizing sgRNA primers targeting the VASA gene promoter; the sgRNA primer is annealed and is subjected to enzyme digestion, and then is connected and inserted into the linearized dCAS9-p300 plasmid, so that the CRISPR/dCAS9-p300 Activator expression plasmid is successfully constructed.
2. The VASA gene reporting vector according to claim 1, wherein the nucleotide sequences of the 5 'and 3' homology arm primers near the VASA gene knock-in site are as follows:
5’homologous arm-F：GCTAGCATCCCATGACTCATCATCTA
5’homologous arm-R：TCATCCAGCCCAGCATTTTG
3’homologous arm-F：GGTACCCCCCTGTATTTATGTCTCACTTGTT
3’homologous arm-R：CTCGAGGATGCAGAGAAGAAAGTAG；
the nucleotide sequence of the EGFP-LoxP-PGK-Puro-LoxP fragment related primer is as follows:
P2A-EGFP-F：
CAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTGTGAGCAAGGGCGAGGAGCT
XHOI-LOXP-PURO-R：
ACTCTCGAGATAACTTCGTATAATGTATGCTATACGAAGTTATGAACCTCTTCGAGGGACCTA
NHEI-P2A-F：
TCAGCTAGCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCA。
3. the VASA gene reporting vector according to claim 1, wherein the gRNA primer is a gRNA3 primer or a gRNA4 primer, and the nucleotide sequences of the gRNA3 primer and the upstream and downstream primers of the gRNA4 primer are as follows:
VASA-gRNA3-F：tttcttggctttatatatcttgtggaaaggacGAAAACAACCTGCAAGTTTG
VASA-gRNA3-R：tcaacttgctatgctgtttccagcatagctctgaaacCAAACTTGCAGGTTGTTTTC
VASA-gRNA4-F：tttcttggctttatatatcttgtggaaaggacgaaacaccGACTCATCATCTACTGGATT
VASA-gRNA4-R：tcaacttgctatgctgtttccagcatagctctgaaacAATCCAGTAGATGATGAGTC。
4. the VASA gene reporting vector according to claim 1, wherein the sgRNA primer is at least one of sgRNA-a and sgRNA-b, and the nucleotide sequences of the sgRNA-a and the sgRNA-b are as follows:
sgRNA-a：TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAACTGTACCACAAACTTGC
sgRNA-b：TCAACTTGCTATGCTGTTTCCAGCATAGCTCTGAAACGCAAGTTTGTGGTACAGTTC。
5. the construction method of the VASA gene report vector is characterized in that the VASA gene report vector comprises a recombinant EGFP-PGK puro-VASA plasmid, a Cas9-gRNA plasmid and a CRISPR dCAs9 activating vector;
(1) The construction method of the recombinant EGFP-PGK puro-VASA plasmid comprises the following steps: designing 5 'and 3' homology arm primers near a VASA gene knock-in site respectively, and amplifying by taking target species genome DNA as a template to obtain homology arm fragments containing enzyme cutting sites, and connecting the homology arm fragments to a pMD-19T vector to obtain a pMD-5&3HA intermediate vector; designing primers by taking TV-hPITX3-Reverse as a template, amplifying by PCR to obtain EGFP-LoxP-PGK-Puro-LoxP fragments, and connecting the EGFP-LoxP fragments with a linearized pMD-5&3HA intermediate vector to obtain recombinant EGFP-PGK Puro-VASA plasmids;
(2) The construction method of the Cas9-gRNA plasmid comprises the following steps: according to the PAM locus, designing and synthesizing a gRNA primer, annealing the synthesized gRNA primer, performing enzyme digestion, and connecting and inserting the synthesized gRNA primer into a linearized pX330 plasmid to obtain a Cas9-gRNA plasmid;
(3) The construction method of the CRISPR dCas9 activation vector comprises the following steps: designing and synthesizing sgRNA primers targeting the VASA gene promoter; the sgRNA primer is annealed and is subjected to enzyme digestion, and then is connected and inserted into the linearized dCAS9-p300 plasmid, so that the CRISPR/dCAS9-p300 Activator expression plasmid is successfully constructed.
6. The method of claim 5, wherein the step of determining the position of the probe is performed,
the nucleotide sequences of the 5 'and 3' homology arm primers near the VASA gene knock-in site are as follows:
5’homologous arm-F：GCTAGCATCCCATGACTCATCATCTA
5’homologous arm-R：TCATCCAGCCCAGCATTTTG
3’homologous arm-F：GGTACCCCCCTGTATTTATGTCTCACTTGTT
3’homologous arm-R：CTCGAGGATGCAGAGAAGAAAGTAG；
the nucleotide sequence of the EGFP-LoxP-PGK-Puro-LoxP fragment related primer is as follows:
P2A-EGFP-F：CAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGAC CTGTGAGCAAGGGCGAGGAGCT
XHOI-LOXP-PURO-R：ACTCTCGAGATAACTTCGTATAATGTATGCTATACGAAGTT ATGAACCTCTTCGAGGGACCTA
NheI-P2A-F：tcaGCTAGCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCA。
7. the method of claim 5, wherein the gRNA primer is a gRNA3 primer or a gRNA4 primer, and the nucleotide sequences of the gRNA3 primer and the upstream and downstream primers of the gRNA4 primer are as follows:
VASA-gRNA3-F：tttcttggctttatatatcttgtggaaaggacGAAAACAACCTGCAAGTTTG
VASA-gRNA3-R：tcaacttgctatgctgtttccagcatagctctgaaacCAAACTTGCAGGTTGTTTTC
VASA-gRNA4-F：tttcttggctttatatatcttgtggaaaggacgaaacaccGACTCATCATCTACTGGATT
VASA-gRNA4-R：tcaacttgctatgctgtttccagcatagctctgaaacAATCCAGTAGATGATGAGTC；
the sgRNA primer is at least one of the sgRNA-a and the sgRNA-b, and the nucleotide sequences of the sgRNA-a and the sgRNA-b are as follows:
sgRNA-a：TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAACTGTAC CACAAACTTGC
sgRNA-b：TCAACTTGCTATGCTGTTTCCAGCATAGCTCTGAAACGCAAGTTTGTGG TACAGTTC。
8. use of the VASA gene reporting vector according to any one of claims 1 to 4 in the following (1) or (2);
(1) Knocking in and activating to express VASA gene;
(2) The VASA gene is studied to participate in the regulation mechanism of sheep germ cell development.
9. A method for knocking-in and activating expression of a VASA gene, characterized in that the VASA gene is knocked-in and activated using the VASA gene reporter vector according to any one of claims 1 to 4.
10. The method of claim 9, wherein the recombinant pEGFP-PGK puro-VASA plasmid and Cas9-gRNA plasmid are co-transfected into target tool cells for puro drug screening, and then the CRISPR dCas9 activating vector is used to transfect the screened cells to activate the expression of the VASA gene in the cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310501774.1A CN116790662A (en) | 2023-05-06 | 2023-05-06 | Construction method and application of sheep VASA gene report vector |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310501774.1A CN116790662A (en) | 2023-05-06 | 2023-05-06 | Construction method and application of sheep VASA gene report vector |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116790662A true CN116790662A (en) | 2023-09-22 |
Family
ID=88047189
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310501774.1A Pending CN116790662A (en) | 2023-05-06 | 2023-05-06 | Construction method and application of sheep VASA gene report vector |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116790662A (en) |
-
2023
- 2023-05-06 CN CN202310501774.1A patent/CN116790662A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yao et al. | Tild-CRISPR allows for efficient and precise gene knockin in mouse and human cells | |
| Zuo et al. | CRISPR/Cas9-mediated targeted chromosome elimination | |
| Sakurai et al. | A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice | |
| CN108660161B (en) | Method for preparing chimeric gene-free knockout animal based on CRISPR/Cas9 technology | |
| CN106191113B (en) | Preparation method of MC3R gene knockout pig | |
| Wang et al. | Delivery of Cas9 protein into mouse zygotes through a series of electroporation dramatically increases the efficiency of model creation | |
| CN105861554B (en) | method for realizing animal sex control based on editing Rbmy gene and application | |
| EP1620452B1 (en) | Treatment of nucleic acid | |
| US11388892B2 (en) | Method for preparing CKO/KI animal model by using CAS9 technology | |
| WO2019206236A1 (en) | Implementation of efficient and precise targeted integration by means of tild-crispr | |
| CN109055434B (en) | Method for correcting pig KIT gene structure mutation by CRISPRCs 9 technology | |
| Iwata et al. | Simple and large-scale chromosomal engineering of mouse zygotes via in vitro and in vivo electroporation | |
| Kang et al. | Apancreatic pigs cloned using Pdx1-disrupted fibroblasts created via TALEN-mediated mutagenesis | |
| CN110938629B (en) | Complete sgRNA for specifically recognizing pig Wip1 gene and application and product thereof | |
| AU2021268263A1 (en) | Methods for improving the health of porcine species by targeted inactivation of CD163 | |
| CN105671045B (en) | A kind of method of sheep embryo fibroblast homologous recombination repair frequency after raising gene editing | |
| CN110862988A (en) | sgRNA, CREBRF dot-pattern-modified Bama miniature pig constructed by sgRNA and application of sgRNA and CREBRF dot-pattern-modified Bama miniature pig | |
| CN111549070B (en) | Method for editing X chromosome multicopy gene to realize animal sex control | |
| CN116790662A (en) | Construction method and application of sheep VASA gene report vector | |
| CN115261360A (en) | Method for constructing gata6 gene knockout zebra fish model | |
| CN114480497A (en) | Construction method and application method of ep400 gene knockout zebra fish heart failure model | |
| EP4144857A1 (en) | Animal preparation method | |
| Tesson et al. | Genome editing in rats using TALE nucleases | |
| JP2023518379A (en) | Optimized methods for cleavage of target sequences | |
| CN114891791B (en) | sgRNA of specific targeting canine Rosa26 gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |