CN113174406A - Preparation method of zebra fish LGP2 gene knockout homozygote - Google Patents
Preparation method of zebra fish LGP2 gene knockout homozygote Download PDFInfo
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Abstract
A preparation method of zebra fish LGP2 gene knockout homozygote comprises determining a zebra fish LGP2 gene knockout gRNA target site, and designing a primer LGP2-1F/R according to the target site; then, a gRNA fragment is obtained by using the designed primer; and then, mixing the ZCAS9mRNA and the gRNA, introducing the mixture into wild zebra fish, and culturing to obtain stably inherited LGP2 gene knockout homozygote zebra fish. Therefore, a knockout homozygote aiming at the zebra fish LGP2 and homologous genes thereof can be successfully constructed, a zebra fish living body with the LGP2 completely lost can be obtained, and a usable model is provided for the systematic and deep research of the functions of the fish LGP 2.
Description
Technical Field
The invention belongs to the field of aquatic products and biological genes, and particularly relates to a preparation method of zebra fish LGP2 gene knockout homozygotes.
Background
The pattern recognition receptor member LGP2 has the functions of recognizing and binding virus nucleic acid RNA, and regulating virus infection immune response, and finally influences the resistance of fish to viruses. However, the current functional research of LGP2 in fish mainly focuses on the reduction or increase of gene expression level to some extent at cellular level, and no functional deletion homozygote for LGP2 knockout of fish living bodies exists, so that the systematic and intensive research and comprehensive evaluation on the function of LGP2 cannot be carried out.
Disclosure of Invention
The invention aims to provide a preparation method of zebra fish LGP2 gene knockout homozygote aiming at the defects of the prior art, which can obtain zebra fish living bodies with complete loss of LGP2 function by successfully constructing knockout homozygote aiming at zebra fish LGP2 and homologous genes thereof, and provide an available model for systematic in-depth research of the LGP2 function of fish; the zebra fish LGP2 knockout homozygous submodel is used for carrying out functional research, so that the immune function exerted by LGP2 and an influenced signal path can be accurately evaluated, and an experimental model is provided for research on the molecular function and interaction mechanism of a fish RLR path.
In order to achieve the purpose, the invention adopts the technical scheme that: a pair of primers is designed according to a gRNA target site sequence knocked out by a zebra fish LGP2 gene, and the primer pair is LGP 2-1F: ataGAGGCTGCTTCCCACCGTTGCgt (SEQ ID NO.2) and LGP 2-1R: taaaacGCAACGGTGGGAAGCAGCCT (SEQ ID NO. 3).
The invention also provides a preparation method of the zebra fish LGP2 gene knockout homozygote, which comprises the following steps:
A. obtaining a gRNA fragment using the above primer (LGP 2-1F/R);
specifically, the gRNA fragment is prepared according to the following steps: (1) determining a gRNA target site knocked out by the zebra fish LGP2 gene, and designing a primer LGP2-1F/R according to the target site; (2) connecting the primer with a gRNA cloning framework to obtain a transcription template, obtaining a gRNA in-vitro transcription template according to the template PCR, and then carrying out in-vitro transcription according to the in-vitro transcription template. Preferably, the gRNA cloning scaffold is pT7-gRNA _ BbsI.
B. And (3) mixing the ZCAS9mRNA and the gRNA, introducing the mixture into wild zebra fish, and culturing to obtain stably inherited LGP2 gene knockout homozygote zebra fish.
Wherein the ZCAS9mRNA is obtained by single-enzyme digestion of a linearized pSP6-2sNLS-ZCAS9 vector by XbaI to obtain an in vitro transcription template of ZCAS9mRNA and performing in vitro transcription according to the template. Wherein, the enzyme cutting system is as follows: ZCAS 920 ug, Buffer 10X 10uL, XbaI 5uL, add RNase-free water to a total reaction volume of 100 uL.
Wherein ZCAS9mRNA and gRNA are mixed according to the ratio of 600pg to 100pg, and the introduction of the wild zebra fish refers to the injection of the wild zebra fish single-cell embryo.
Specifically, the process of obtaining stably inherited LGP2 gene knockout homozygous zebra fish by the culture is as follows: (1) carrying out gene sequencing on the injected zebra fish to obtain an LGP2 gene knockout zebra fish heterozygote F0 generation; (2) crossing the F0 generation with AB type wild zebra fish to obtain F1 generation, and performing gene sequencing to obtain "-6 +1 bp" type F1 generation zebra fish; (3) carrying out internal crossing on mature F1 generations of female and male heterozygotes of "-6 +1 bp" type to obtain F2 generations, and carrying out gene sequencing to obtain female homozygote zebrafish of "-6 +1 bp" gene editing F2 generations and male heterozygote zebrafish of "-6 +1 bp" gene editing F2 generations; (4) mating female homozygote zebrafish edited by the gene of "-6 +1 bp" F2 generation with male heterozygote zebrafish edited by the gene of "-6 +1 bp" F2 generation to generate F3 generation, and obtaining F3 "-6 +1 bp" type female and male homozygote zebrafish after gene sequencing; (5) and (3) carrying out internal crossing on the F3 '-6 +1 bp' type female homozygous zebra fish and the F3 '-6 +1 bp' type male homozygous zebra fish to obtain the LGP2 gene knockout homozygous zebra fish. Wherein, the primer adopted in the gene sequencing is LGP 2-JF: 5'-CTGGTGGTGATCAGTGAAAC-3' (SEQ ID NO.4) and LGP 2-JR: 5'-CACCTAGTACACAAAACACA-3' (SEQ ID NO. 5).
The invention has the following beneficial effects:
(1) and a gRNA target point and a corresponding primer sequence (LGP2-1F/R) which can knock out the zebra fish LGP2 gene and the homologous isomer thereof are obtained by screening.
(2) Based on AB type wild zebra fish, the homozygous zebra fish with LGP2 knockout is successfully obtained for the first time.
(3) By comparing the expression changes of the homozygote, the wild zebra fish RLR passage member and the interferon passage member mediated by the same, LGP2 is found to negatively regulate the antiviral response of interferon, and a high-quality model is provided for the systematic and deep research of the functions of LGP2 and other RLR members participating in the virus infection process.
Drawings
FIG. 1 is a zebrafish LGP2 "-6 +1 bp" type knockout sequencing assay.
FIG. 2 is a graph of the effect of zebrafish LGP2 knockout on RLR membership and interferon response pathways. (the ordinate represents the relative expression level of the gene)
Wherein A is directed to ifnphi1, ifnphi2, ifnphi4, ifih1, mxa, and B is directed to ifnphi3, mavs, ddx58, mxb.
Detailed Description
Example 1
In the experiment, a specific primer for knocking out the LGP2 gene of the zebra fish is designed based on CHOPCHOP software, and the gene knocking out experiment of LGP2 is carried out on AB type wild zebra fish embryos.
1. Screening and preparation of gRNA target spot for knockout of zebra fish LGP2
LGP2 gene fragment CCTAGGTCCTGTATAGCAGACATACATATATAAACAAGTCGTTTGAAAACTAATTTATGTTGTACCATTTCTCTCATTCTCAGATCTGTGCCAATCTGGATTCAAAAATTGTGTCCACCAAGAATTACACACCGATGCTGCAGAACTTTGTGCCAAAACCCAAAAAGGAATACGACATTGTCGAAAGAAGAGATAAAGTACTGTCTTTCTGCCATGCAAATCCCAGACCTGCTGCTTGATCATTCAAGACCAATGTTTATGTATGTTTATTTGTGTGCAACAGGATCCATTTGGTGACCACTTGAAGTCAATGATGTTAATGATTCATGAGTTTATGCCAGCAACGGTGGGAAGCAGCCTGAGAGAACTAGGCACCCAGGAATATGAGGCTGATGTGGTGGAACTGGAGAAAGCAGGTTGGAGGTCATTTTTACTAGTTTGAATTTTACATTCAGAAATCTATGCAAAGTTTACTGGGTTTAATGGTGTGTTTTGGGAAACTAGGGTGA (SEQ ID NO.1), wherein the underlined region is located in exon 5 of LGP2 gene, belongs to a common conserved sequence of zebrafish LGP2 and homologous genes thereof, and is a selected zebrafish LGP2 gene knockout gRNA target site. Designing a gene knockout primer aiming at the target site, wherein the designed target site primer sequence is as follows:
LGP2-1F：ataGAGGCTGCTTCCCACCGTTGCgt(SEQ ID NO.2)，
LGP2-1R：taaaacGCAACGGTGGGAAGCAGCCT(SEQ ID NO.3)。
the above-mentioned each set of oligo sequences was applied with ddH2O was dissolved in a 10. mu.M solution, annealed (annealing procedure: heating at 95 ℃ for 5min, then lowering 1 ℃ every 30s to 4 ℃) to obtain sticky end small fragments. The annealed sticky end small fragment was ligated to the gRNA cloning backbone pT7-gRNA _ BbsI, with the linker being 0.5. mu.L of pT7-gRNA _ BbsI, 1. mu.L of each set of the annealed sticky end small fragments, 2 XSolution I (Takara) 5. mu.L, plus ddH2O3.5 μ L; 1h at 16 ℃. Transferring the ligation product into Escherichia coli BL21, picking single clone for sequencing, selecting clone plasmid with correct sequence as transcription template (namingpT7gRNA plasmid). A gRNA in-vitro transcription template is obtained by amplifying the conventional primer T7gRNA-F/R and pT7gRNA plasmid 5ng under the conditions of 95 ℃ for 15s, 60 ℃ for 20s and 72 ℃ for 15s in 45 cycles by using a high fidelity enzyme Prime Star PCR. According to Ambion
The kit completes in vitro transcription of the gRNA. mirVana according to AmbionTMThe miRNA separation kit completes the in vitro transcription, purification and recovery of gRNA.
zCas9mRNA preparation:
preparation of in vitro transcription template for zCas9 mRNA: the pSP6-2sNLS-ZCAS9 vector is linearized by using an XbaI single enzyme, wherein the enzyme digestion system is as follows: ZCAS 920 ug, Buffer 10X 10uL, XbaI 5uL, add RNase-free water to a total reaction volume of 100uL, react at 37 ℃ for 2 hours. Agarose gel electrophoresis confirmed the linearization was complete and the linearized product was recovered and transcribed in vitro for 2h at 37 ℃ to obtain zcAS9 mRNA.
3. Design of sequencing primers
In order to detect the gene knockout effect, a pair of sequencing detection primers is designed as follows: LGP 2-JF: 5'-CTGGTGGTGATCAGTGAAAC-3' (SEQ ID NO.4) and LGP 2-JR: 5'-CACCTAGTACACAAAACACA-3' (SEQ ID NO.5), corresponding to an amplified PCR product of 509 bp. The 509bp product amplification region contains a target region and can be used for detecting whether the target region is knocked out.
4. Verifying microinjection and cutting efficiency;
wild type zebrafish single cell stage embryos were injected according to the zCas9mRNA 600pg/gRNA100pg mixing ratio, 150 embryos per target. 20 juvenile fishes randomly selected 2 days after injection are subjected to genome PCR sequencing, and the result shows that a target point corresponding to a primer LGP2-1F/R has a random peak, which indicates that a knockout heterozygote (F0 generation) is successfully obtained.
Editing zebrafish screening available in generations F1 and F2
Carrying out outcrossing on the zebra fish of the F0 generation and AB type wild zebra fish to obtain zebra fish of the F1 generation; aiming at each F0 zebra fish, correspondingly and randomly selecting 10 fertilized eggs of F1 generation fishes which are generated and normally develop, extracting DNA after cracking, carrying out PCR sequencing detection, selecting fragments with double peaks at target spots to carry out TA cloning identification cutting mode, and displaying the result that the positive rate of the F1 generation juvenile fishes with the random peaks is 3/9.
The screening results of the zebra fish of the F1 generation after being treated by LGP2-1F/R are as follows: the adult fish tail-shearing positive rate is 13/22, and the adult fish tail-shearing positive rate is 4 pieces of "-85 bp", 5 pieces of "-6 +1 bp", 1 piece of "-5 bp", and the rest is wild type fish. Carrying out internal crossing on the-6 +1bp type female and male heterozygotes in the mature F1 generation to obtain an F2 generation. The 12-tailed F2 zebra fish is subjected to tail shearing, DNA extraction and PCR sequencing detection, and 2 male heterozygous fish, 3 female heterozygous fish and 1 female homozygous fish of the type of "-6 +1 bp" are found.
Screening and identification of LGP2 "-6 +1 bp" -type knockout homozygote
Mating the female homozygote zebrafish edited by the gene of "-6 +1 bp" F2 with the male heterozygote zebrafish edited by the gene of "-6 +1 bp" F2 to generate an F3 generation fish; cutting tails one by one, extracting-6 +1bp type F3 generation fish tail DNA, amplifying a target region by PCR, and sequencing to obtain 3-6 +1bp type female homozygotes, male homozygotes and 3 heterozygotes. The female and male homozygous zebra fish of F3 "-6 +1 bp" type was subjected to internal crossing to obtain 60F 4, F4 were all "-6 +1 bp" type homozygous zebra fish (FIG. 1).
Example 2 Gene knockout homozygote LGP2 pathway Gene expression assay
60 7-day-long-term-born zebra fish are randomly selected from F4 '-6 +1 bp' type gene knockout homozygote zebra fish for LGP2 signal channel member gene expression detection, and the results are combined with the results shown in FIG. 2, so that the LGP2 knockout homozygote is up-regulated in gene expression and down-regulated in gene expression of mxb compared with AB type wild zebra fish ifih1, ddx58, ifnphi1, ifnphi2, ifnphi3, ifnphi4, mxa and mavs; the fact that the knockout of the zebrafish LGP2 gene has a substantial influence on the RLR members and the interferon response channels mediated by the RLR members is shown, and LGP2 negatively regulates the RLR channel members ifih1 and ddx58 and the interferon antiviral responses mediated by the RLR channel members.
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Claims (10)
1. A pair of primers, characterized in that the pair of primers is
LGP2-1F, the sequence of which is shown in SEQ ID NO.2,
LGP2-1R, the sequence is shown in SEQ ID NO. 3.
2. A preparation method of zebra fish LGP2 gene knockout homozygote is characterized by comprising the following steps:
A. obtaining a gRNA fragment using the primer of claim 1;
B. and (3) mixing the ZCAS9mRNA and the gRNA, introducing the mixture into wild zebra fish, and culturing to obtain stably inherited LGP2 gene knockout homozygote zebra fish.
3. The method of claim 2, wherein the gRNA fragment of step a is prepared by the following steps:
(1) determining a gRNA target site of knockout of a zebra fish LGP2 gene, and designing a primer LGP2-1F/R according to the target site, wherein the primer LGP2-1F/R is the primer of claim 1;
(2) connecting the primer with the gRNA cloning framework to obtain a transcription template, obtaining a gRNA in-vitro transcription template according to the template PCR, and then carrying out in-vitro transcription.
4. The method for preparing a zebrafish LGP2 gene knockout homozygote according to claim 3, wherein the backbone of the gRNA clone is pT7-gRNA _ BbsI.
5. The preparation method of the zebrafish LGP2 gene knockout homozygote as claimed in claim 2, wherein the ZCAS9mRNA in the step B is obtained by single-enzyme digestion of a linearized pSP6-2sNLS-ZCAS9 vector by XbaI, and an in vitro transcription template of the ZCAS9mRNA is obtained, and then the in vitro transcription is carried out according to the template.
6. The method for preparing the zebrafish LGP2 gene knockout homozygote of claim 5, wherein the enzyme digestion system is: ZCAS 920 ug, Buffer 10X 10uL, XbaI 5uL, add RNase-free water to a total reaction volume of 100 uL.
7. The method for preparing the zebrafish LGP2 gene knockout homozygote of claim 2, wherein in step B the ZCAS9mRNA and the gRNA are mixed at a ratio of 600pg:100 pg.
8. The method for producing a knockout homozygote of LGP2 of zebrafish according to claim 2, wherein the introduction of wild-type zebrafish is performed by injecting embryo at single cell stage of wild-type zebrafish.
9. The method for producing a zebrafish LGP2 knockout homozygote according to claim 2, wherein the culturing in step B provides stably inherited LGP2 knockout homozygote zebrafish as follows:
(1) carrying out gene sequencing on the injected zebra fish to obtain an LGP2 gene knockout zebra fish heterozygote F0 generation;
(2) crossing the F0 generation with AB type wild zebra fish to obtain F1 generation, and performing gene sequencing to obtain "-6 +1 bp" type F1 generation zebra fish;
(3) carrying out internal crossing on mature F1 generations of female and male heterozygotes of "-6 +1 bp" type to obtain F2 generations, and carrying out gene sequencing to obtain female homozygote zebrafish of "-6 +1 bp" gene editing F2 generations and male heterozygote zebrafish of "-6 +1 bp" gene editing F2 generations;
(4) mating female homozygote zebrafish edited by the gene of "-6 +1 bp" F2 generation with male heterozygote zebrafish edited by the gene of "-6 +1 bp" F2 generation to generate F3 generation, and obtaining F3 "-6 +1 bp" type female and male homozygote zebrafish after gene sequencing;
(5) and (3) carrying out internal crossing on the F3 '-6 +1 bp' type female homozygous zebra fish and the F3 '-6 +1 bp' type male homozygous zebra fish to obtain the LGP2 gene knockout homozygous zebra fish.
10. The method for preparing the zebrafish LGP2 gene knockout homozygote of claim 9, wherein the primer used for gene sequencing is LGP2-JF/JR, and the sequences are shown as SEQ ID No.4 and SEQ ID No.5, respectively.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110498783.0A CN113174406A (en) | 2021-05-08 | 2021-05-08 | Preparation method of zebra fish LGP2 gene knockout homozygote |
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| CN113416752A (en) * | 2021-06-23 | 2021-09-21 | 周娟 | Mog1 gene knockout zebra fish model and application |
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