CN111269936B - Plasmid vector set, DNA set, and method for editing foreign gene introduced gene - Google Patents
Plasmid vector set, DNA set, and method for editing foreign gene introduced gene Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
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- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
Abstract
The invention belongs to the technical field of molecular precise improvement breeding of genetic engineering and poultry varieties, and discloses a gene editing method for introducing plasmid vector groups, DNA groups and exogenous genes, which comprises the following steps: the P1 plasmid, the P2 plasmid, and the P3 plasmid; the DNA module comprises: RNA-mediated Cas9 endogenous endonuclease gene, specific site-mediated sgRNA gene, and homologous DNA fragments specific for the site and insertion site of the introduced gene fragment. The method of the present invention, which is capable of integrating a DNA fragment of interest into a vector set at a suitable location in the genome of a poultry organism, facilitates the insertion of the DNA fragment of interest into a suitable location in the genome of an animal cell, thereby obtaining a transgenic or chimeric animal individual. The vector group disclosed by the invention has the characteristics of easiness in construction, easiness in batch production, safety and high efficiency.
Description
Technical Field
The invention belongs to the technical field of molecular precise improvement breeding of genetic engineering and poultry varieties, and particularly relates to a gene editing method for plasmid vector group, DNA group and exogenous gene introduction.
Background
The laying fowl industry is a major characteristic poultry industry in China, particularly the laying duck industry, so that related breeding research is more abroad, but the hatching production of the male poultry in the hatching process is the largest resource waste in the development of the laying fowl industry because the use proportion of the male poultry is extremely low (the proportion of the male poultry to the female poultry is only 1: 20-30 under the condition of artificial insemination), and the problem of how to utilize the latest gene editing technology to introduce exogenous genes to mark fertilized eggs of the male poultry is a problem to be solved urgently in the development of the laying fowl industry at present. The current gene editing technology has great significance for gene function research, drug screening and gene therapy. The transgenic operation of poultry to obtain poultry eggs with high added value is also a hot spot of the current egg poultry industry breeding research. However, the fertilization process of poultry eggs occurs in the oviduct, which is earlier than the production process, and rapid embryonic development occurs in a short time after fertilization, and the transgenic operation of multicellular embryos is very complicated, which has very strict requirements on the time of the transgenic operation. Meanwhile, the traditional operation needs lentivirus mediation, and the lentivirus-mediated foreign gene introduction has the following defects: first, lentivirus introduction is performed by using a random recombination mechanism of LTR regions of a virus plasmid, so that introduction randomness exists, namely, the position of introduced foreign genes is uncertain, and poultry mutation can be caused by the introduction position; secondly, the lentivirus is an experimental biological tool material modified from the HIV, although the toxic gene of the lentivirus is inactivated to a great extent, the operation has great harmfulness and the operation process needs to be in contact with the lentivirus, so that the lentivirus has certain harmfulness to operators; thirdly, the lentivirus transfection has complex biological steps of virus packaging, cell inoculation and the like, the method is too complex to operate, and common technicians cannot complete the operation by needing certain biotechnological operation experience, so that the application of the exogenous gene introduction method in the poultry industry has certain inconvenience. Therefore, the conventional exogenous gene introduction method cannot meet the current requirements for the exogenous gene introduction in the rapid and accurate breeding improvement technology, and many researchers continuously explore new and more effective breeding improvement methods for exogenous gene introduction to overcome the defects of the conventional exogenous gene introduction method for breeding improvement.
In summary, the problems of the prior art are as follows: due to the common problems of the conventional lentivirus transfection, the introduction of lentiviruses in the traditional transgenic operation has randomness and the position of introduced foreign genes is uncertain, and the introduced position causes poultry variation; the operation of the lentivirus has great harmfulness, and the operation process needs to be contacted with the virus, so that the operation personnel has certain harmfulness; the operation is too complicated.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a plasmid vector group, a DNA group and a gene editing method for introducing exogenous genes.
The present invention is achieved by a plasmid vector set comprising: the P1 plasmid, the P2 plasmid, and the P3 plasmid;
the P1 plasmid comprises an RNA-mediated Cas9 endogenous endonuclease gene;
the P2 plasmid contains a specific site-mediated sgRNA gene;
the P3 plasmid contains the homologous DNA fragment specific to the introduced gene fragment and the insertion site.
Further, the P3 plasmid contained two homologous DNA fragments flanking the insertion site.
Furthermore, the foreign gene segment to be introduced is arranged between two homologous DNA segments in the P3 plasmid, wherein the homologous DNA segments are specific to the poultry genome insertion site.
Further, the P2 plasmid contains a specific site fragment homologous to the poultry genome and directs the action of Cas9 endogenous nuclease cleavage at this site.
Further, the P3 plasmid inserts the introduced gene segment into the genome of poultry tissue at a specific site by homologous complementation under the mediation of sperm or fertilized egg blastoderm.
It is another object of the present invention to provide a DNA module constructed using the plasmid vector set, the DNA module comprising: an RNA-mediated Cas9 endogenous endonuclease gene, a specific site-mediated sgRNA gene, and a homologous DNA fragment specific to the site and insertion site of the introduced gene fragment.
Another object of the present invention is to provide a gene editing method for foreign gene introduction using the DNA module, the method comprising the steps of:
inserting a required DNA assembly into a plasmid vector through restriction enzymes and ligases, or directly synthesizing long-chain DNA;
step two, semen collected by a male individual is mixed with the plasmid vector group-liposome 2000 compound, and artificial fertilization is carried out on the female individual;
and step three, after the fertilized eggs are incubated for 48 hours, opening a diameter opening at the middle position of the eggshell closest to the blastoderm, injecting the plasmid vector into the lower cavity of the embryonic blastoderm at the development stage through a capillary glass tube, filling sterile RPMI 1640 culture solution into the cavity, adding antibiotics, sealing by using a medical wound plaster, and incubating.
The invention also aims to provide the laying duck bred by the gene editing method for introducing the exogenous gene.
Another object of the present invention is to provide poultry bred by the gene editing method for foreign gene introduction.
The semen-mediated method directly delivers the plasmid vector group with the target DNA segment into the egg cells of the female individual and integrates the plasmid vector group into the genome of the egg cells, so that homozygote transgenic individuals can be obtained at one time; the blastoderm microinjection method mostly generates chimera transgenic individuals, and homozygote transgenic individuals are obtained within three generations by screening heritable germ line chimeras; different target DNA fragments are selected according to the production purpose of the transgenic animal, and the method can be applied to the aspects of gene function analysis, breed improvement, human disease model animal manufacture, donor animal manufacture for organ transplantation, production of physiologically active substances and the like.
The method of the present invention, which is capable of integrating a DNA fragment of interest into a vector set at a suitable location in the genome of a poultry organism, facilitates the insertion of a DNA fragment of interest into a suitable location in the genome of an animal cell, thereby obtaining a transgenic or chimeric animal individual. The vector group disclosed by the invention has the characteristics of easiness in construction, easiness in batch production, safety and high efficiency.
Drawings
FIG. 1 is a flowchart of a gene editing method for foreign gene introduction according to an embodiment of the present invention.
FIG. 2 is a schematic diagram of the P1 plasmid vector provided by the embodiment of the present invention.
FIG. 3 is a schematic diagram of the P2 plasmid vector provided by the embodiment of the present invention.
FIG. 4 is a schematic diagram of a P3 plasmid vector provided by the embodiment of the invention.
FIG. 5 is a schematic diagram of a process for preparing a plasmid vector set mixture according to an embodiment of the present invention.
FIG. 6 is a schematic diagram of a process for introducing a target gene by semen-mediated delivery according to an embodiment of the present invention.
FIG. 7 shows the results of detection of fluorescence gene introduced into plasmid (M is marker; NS is negative individual detection result; PS is positive individual detection result) according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The current gene editing technology has great significance for gene function research, drug screening and gene therapy. Poultry eggs with high added value are obtained by carrying out transgenic operation on poultry.
The following detailed description of the principles of the invention is provided in connection with the accompanying drawings.
The plasmid vector group provided by the embodiment of the invention comprises: one plasmid containing the RNA-mediated Cas9 endogenous endonuclease gene P1, one plasmid containing the specific site-mediated sgRNA gene P2, and one plasmid containing the homologous DNA fragment P3 specific for the introduced gene fragment and the insertion site. Wherein the P3 plasmid can insert the introduced gene segment into the specific site in the poultry tissue genome through homologous complementation under the mediation of sperms or fertilized egg blastoderms.
The P3 plasmid contains two homologous DNA fragments flanking the insertion site.
The middle of two homologous DNA fragments in the P3 plasmid, which are specific to the insertion site of the poultry genome, is the exogenous gene fragment to be introduced.
The P2 plasmid contains a specific site fragment homologous to the poultry genome and is capable of directing the action of Cas9 endogenous nucleases to cleave there.
The DNA assembly provided by the embodiment of the invention comprises: (P1) comprising an RNA-mediated Cas9 endogenous endonuclease gene; (P2) specific site-mediated sgRNA gene; (P3) contains homologous DNA fragments (homo log Part and Right homo log Part) specific to the site of introduction of the gene fragment (P4) and the insertion site.
The P2 plasmid contains a specific site fragment sgRNA homologous to the poultry genome, the sequence of which is SEQ ID NO: 1: 5'-tgtgaacttt tgccaaattt-3' are provided.
The Left Homolog Part sequence in P3 is SEQ ID NO: 2: 5'-gttttcttat aaggaaggga aaa-3', Right Homolog Part sequence of SEQ ID NO: 3: 5'-aaccttgatt tagaaccaac ttg-3'; in the middle of Left Homolog Part and Right Homolog Part in the P3 plasmid are the foreign expression gene fragment to be introduced and the universal promoter CMV.
As shown in fig. 1, the method for editing a gene into which a foreign gene is introduced according to an embodiment of the present invention includes the following steps:
s101: constructing a vector group: the required DNA components can be inserted into a plasmid vector by restriction enzymes and ligases, or the synthesis of long-chain DNA can be directly carried out;
s102: introduction of genes of interest by seminal fluid mediation: semen collected from a male individual is mixed with the plasmid vector set-liposome 2000 complex, and then the mixture is used to artificially fertilize a female individual;
s103: the target gene is introduced by microinjection technology: after 48 hours of incubation of the fertilized eggs, an opening with the diameter of about 1cm is opened at the position closest to the middle position of the eggshell of the blastoderm, plasmid vectors with proper volume and concentration are injected into the lower cavity of the embryonic blastoderm at the development stage through a capillary glass tube, the cavity is filled with sterile RPMI 1640 culture solution, and after antibiotics (ampicillin and streptomycin) are added, the eggs are sealed by a medical adhesive bandage and incubated.
The gene editing method for introducing the exogenous gene provided by the embodiment of the invention comprises three key components: one containing the RNA-mediated Cas9 endogenous endonuclease gene P1 plasmid, one containing the specific site-mediated sgRNA gene P2 plasmid, and one containing the introduced promoter, gene expression fragments, and specific homologous DNA fragments (Homolog Part and Right Homolog Part) flanking the insertion site P3 plasmid. Mixing three plasmids of P1, P2 and P3 according to the molecular number and the proportion of 1:1:1 to prepare a plasmid vector group mixed solution; preparing a compound mixed solution from semen collected from a male individual, a plasmid vector group and a liposome 2000 reagent, then using the mixed solution to perform artificial fertilization on a female individual, and collecting a poultry individual which contains an exogenous gene and is hatched from a fertilized egg; or collecting fertilized eggs, incubating for 48 hours, opening an opening with the diameter of about 1cm at the middle position of the eggshell closest to the blastoderm, injecting the mixed liquid of the plasmid vector group into the lower cavity of the embryonic blastoderm at the development stage through a capillary glass tube, filling the cavity with sterile RPMI 1640 culture solution, adding antibiotics (ampicillin and streptomycin), sealing with a medical wound bandage, and continuing to incubate poultry individuals containing exogenous genes.
The application of the principles of the present invention will now be described in further detail with reference to specific embodiments.
Example 1: a method for inserting exogenous GFP fluorescence into a specific locus of a duck genome by using Cas9-HDR and sperm (or fertilized blastoderm) mediation is designed, and comprises the following steps:
(1) constructing a vector group: the vector set comprises three key components: one containing the RNA-mediated Cas9 endogenous endonuclease gene P1 plasmid, one containing the specific site-mediated sgRNA gene P2 plasmid, and one containing the introduced promoter, GFP fluorescent gene expression fragment, and specific homologous DNA fragments (Homolog Part and Right Homolog Part) flanking the insertion site P3 plasmid. Synthesizing long-chain DNA (deoxyribonucleic acid) P3, and mixing three plasmids P1, P2 and P3 according to the molecular number and the proportion of 1:1:1 to prepare a plasmid vector group mixed solution.
(2) Introduction of genes of interest by seminal fluid mediation: semen collected from a male duck individual is mixed with a plasmid vector group mixed solution and a liposome 2000 reagent to prepare a compound mixed solution, then the compound mixed solution is used for artificial fertilization of a female duck individual, and a poultry individual containing a GFP fluorescent gene is collected and hatched from a fertilized egg.
(3) Introducing a target gene through the mediation of fertilized duck placenta: after collecting fertilized eggs and incubating for 48 hours, opening an opening with the diameter of about 1cm at the middle position of an eggshell closest to a blastoderm, injecting a plasmid vector group mixed solution containing GFP fluorescent genes into the lower cavity of the blastoderm of the embryo at the development stage through a capillary glass tube, filling sterile RPMI 1640 culture solution into the cavity, adding antibiotics (ampicillin and streptomycin, namely a dual-resistance reagent), sealing the cavity by using a medical wound plaster, and continuously incubating duck individuals containing exogenous GRP fluorescent genes.
Example 2: a method for inserting exogenous chicken OVL gene into specific locus of duck genome by using Cas9-HDR and sperm (or fertilized blastoderm) mediation is designed, comprising the following steps:
(1) constructing a vector group: the vector set comprises three key components: one containing the RNA-mediated Cas9 endogenous endonuclease gene P1 plasmid, one containing the specific site-mediated sgRNA gene P2 plasmid, and one containing the introduced promoter, the OVL gene expression fragment, and the specific homologous DNA fragments (Homolog Part and Right Homolog Part) flanking the insertion site. Synthesizing long-chain DNA (deoxyribonucleic acid) P3, and mixing three plasmids P1, P2 and P3 according to the molecular number and the proportion of 1:1:1 to prepare a plasmid vector group mixed solution.
(2) Introduction of genes of interest by seminal fluid mediation: semen collected from a male duck individual, the mixed solution of the plasmid vector group and the liposome 2000 reagent are prepared into a compound mixed solution, then the compound mixed solution is used for carrying out artificial fertilization on a female duck individual, and the duck individual containing the chicken OVL gene is hatched by collecting fertilized eggs.
(3) Introduction of genes of interest mediated by fertilized placenta: after the fertilized eggs are collected and incubated for 48 hours, an opening with the diameter of about 1cm is opened at the middle position of the eggshell which is closest to the blastoderm, the mixed solution of the plasmid vector group containing the chicken OVL gene is injected into the lower cavity of the embryonic blastoderm at the development stage through a capillary glass tube, the cavity is filled with sterile RPMI 1640 culture solution, antibiotics (ampicillin and streptomycin, namely, double-resistant reagents) are added, then the medical wound plaster is used for sealing, and the duck individuals containing the exogenous OVL gene are continuously incubated.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the present invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive efforts by those skilled in the art based on the technical solution of the present invention.
Sequence listing
<110> Zhejiang province academy of agricultural sciences
<120> plasmid vector set, DNA set, and method for gene editing by foreign gene introduction
<141> 2017-12-29
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gttttcttat aaggaaggga aaa 23
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aaccttgatt tagaaccaac ttg 23
Claims (2)
1. A plasmid vector set, comprising: the P1 plasmid, the P2 plasmid, and the P3 plasmid;
the P1 plasmid comprises an RNA-mediated Cas9 endogenous endonuclease gene;
the P2 plasmid contains a specific site-mediated sgRNA gene;
the P3 plasmid contains an introduced gene segment and a homologous DNA segment specific to an insertion site;
the sequence of the P2 plasmid is SEQ ID NO: 1;
the Left homo log Part sequence of P3 is SEQ ID NO: 2; the sequence of RightHomolog Part is SEQ ID NO: 3;
the P3 plasmid contained two homologous DNA fragments flanking the insertion site;
the middle of two homologous DNA fragments in the P3 plasmid, which are specific to the poultry genome insertion site, is an exogenous gene fragment to be introduced;
the P2 plasmid contains a specific site fragment homologous to the poultry genome and guides the action of Cas9 endogenous nuclease cleavage at the site;
the P3 plasmid inserts the introduced gene segment into the specific site in the poultry tissue genome through homologous complementation under the mediation effect of sperms or fertilized egg blastodiscs;
the DNA assembly comprises: RNA-mediated Cas9 endogenous endonuclease gene, specific site-mediated sgRNA gene, and homologous DNA fragments specific for the site and insertion site of the introduced gene fragment.
2. A gene editing method for foreign gene introduction using the DNA module of claim 1, comprising the steps of:
inserting a required DNA assembly into a plasmid vector through restriction endonuclease and ligase, or directly synthesizing long-chain DNA;
mixing semen collected by the male individual with the plasmid vector group-liposome 2000 compound, and performing artificial fertilization on the female individual;
and step three, after the fertilized eggs are incubated for 48 hours, opening a diameter opening at the middle position of the eggshell closest to the blastoderm, injecting the plasmid vector into the lower cavity of the embryonic blastoderm at the development stage through a capillary glass tube, filling sterile RPMI 1640 culture solution into the cavity, adding antibiotics, sealing by using a medical wound plaster, and incubating.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805118A (en) * | 2015-04-22 | 2015-07-29 | 扬州大学 | Method for targeted knockout of specific gene of Suqin yellow chicken embryonic stem cell |
| CN105296537A (en) * | 2015-12-12 | 2016-02-03 | 西南大学 | Fixed-point gene editing method based on intratestis injection |
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2017
- 2017-12-29 CN CN201711484426.9A patent/CN111269936B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805118A (en) * | 2015-04-22 | 2015-07-29 | 扬州大学 | Method for targeted knockout of specific gene of Suqin yellow chicken embryonic stem cell |
| CN105296537A (en) * | 2015-12-12 | 2016-02-03 | 西南大学 | Fixed-point gene editing method based on intratestis injection |
Non-Patent Citations (3)
| Title |
|---|
| CRISPR mediated somatic cell genome engineering in the chicken;Nadège Véron 等;《Developmental Biology》;20150813;第407卷;68-74 * |
| CRISPR/Cas9系统在鸡MSTN上的效率验证;胡曼 等;《中国家禽》;20160731;第38卷(第07期);5-9 * |
| 鸡DMRT1基因CRISPR/Cas9载体构建及打靶效率的检测;杨秀荣 等;《中国家禽》;20170228;第39卷(第02期);6-9 * |
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