CN106636201A - MC1R (melanocortin 1 receptor) gene carrier and construction method thereof - Google Patents
MC1R (melanocortin 1 receptor) gene carrier and construction method thereof Download PDFInfo
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Abstract
The invention discloses an MCIR (MelanoCortin 1 Receptor) gene carrier and a construction method thereof. The MCIR gene carrier comprises MC1R-neo and MC1R-GFP (Green Fluorescent Protein), wherein the nucleotide sequence of the MC1R-neo is SEQ ID NO:1, and the nucleotide sequence of the MC1R-GFP is SEQ ID NO:2. A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system is adopted to construct an MC1R gene knock-out vector, and only a sgRNA (Ribonucleic Acid) of which the length is about 20bp needs to be designed by aiming at a gene knock-out point to be connected with a universal Cas9 gene. Therefore, compared with traditional gene knock-out technologies including ZFNs (Zinc finger nuclease), TALENs (Transcription Activator-Like Effector Nucleases) and the like, the construction method disclosed by the invention adopting the CRISPR/Cas9 to construct the gene knock-out vector is simpler and more convenient and is more convenient to be further popularized and applied to subsequent experiments.
Description
Technical field
The invention belongs to genophore technical field, more particularly to a kind of MC1R genophores and its construction method.
Background technology
The hair color or plumage color of animal as an important economic characters, it is determined that sending out in terms of variety and affiliation
Wave important effect.Research finds why different animal hair color or plumage color be, are because that melanic species is different with distribution
Cause.Melanocortin receptor 1 (melanocortin 1 recptor, MC1R), is to control the master that Animal melanin is formed
Gene is wanted, is made up of an extron, expressed in melanocyte, interacted with native ligand a-MSH, by by G eggs
Adjusting a series of cascade reactions in melanocyte, final Jing DOPA or DOPA chromium are closed the cAMP signal paths of white coupling
It is pheomelanin cell and eumelanin into respective derivative, so as to determine the hair color of animal.However, at present on birds, closing
Affect the molecule mechanism of melanin deposition to be still not clear in MC1R, in terms of its gene function, with regard to MC1R gene knockouts and
There is not been reported for the correlative studys such as the impact after knockout to plumage color proterties.CRISPR/Cas9 systems are that bacterium is long-term in bacteriophage
Selection pressure under evolve a kind of one of the immunologic mechanism for effectively resisting foreign DNA invasion for coming.In thalline, CRISPR clusters
PrecrRNA is transcribed under the regulation and control of its leader, and ripe crRNA is processed into the case where tracrRNA and Cas9 is participated in, drawn
Lead the identification of crRNA/tracrRNA/Cas9 complexs and combine foreign DNA particular sequence, DNA double chain is sheared, so as to silence external source base
The expression of cause.CRISPR/Cas9 systems have been developed to a kind of new gene targeting system.Relative to RNAi, ZFN earlier
With TALEN systems, this new targeting system have simple to operate, low cost, efficiency high, can simultaneously silence any amount gene
The advantages of.
At present on birds, the molecule mechanism of melanin deposition is affected to be still not clear with regard to MC1R, in its gene function
Aspect, with regard to correlative studys such as the impacts after MC1R gene knockouts and knockout to plumage color proterties, there is not been reported.
The content of the invention
It is an object of the invention to provide a kind of MC1R genophores and its construction method, it is intended to for family chicken MC1R gene
Knockout is oriented, is that the feather hair color to family chicken carries out artificial regulatory and lays the foundation.
The present invention is achieved in that a kind of MC1R genophores, and the gene orientation that can be used for family chicken MC1R is knocked out, and is suitable for
In scientific research fields such as chicken plumage colors, the artificial control of chicken plumage color proterties is further applicable to, the MC1R genophores include
MC1R-neo and MC1R-GFP；The MC1R-neo nucleotides sequences are classified as SEQ ID NO：1；The MC1R-GFP nucleotide sequences
For SEQ ID NO：2.
Another object of the present invention is to provide a kind of construction method of the MC1R genophores, the construction method bag
Include following steps：
Step one, primer annealing：1ul F-Oligo (100uM), 1ul R-Oligo (100uM), 8ul YSY oligo are moved back
Fiery buffer solution, above solution is mixed in PCR pipe, and gradually 22 DEG C are down to from 95 DEG C with 1.5 DEG C per minute in PCR instrument；
Step 2, connection：0.5ul annealed products, 1ul YSY linearize three-in-one CRISPR/Cas9n plasmids, 1ul T4
Ligase, 2ul 5*T4Buffer, 5.5ul Milli Q；
Step 3, conversion：Pfu >=10 are used after room temperature 15min8Escherichia coli DH5a competent cell converted；
Step 4, transformant checking：Picking monoclonal carries out the checking of 10ul systems bacterium solution PCR after conversion coated plate；
Step 5, is sequenced Jing after PCR Preliminary Identifications；
Step 6, plasmid extraction：37 DEG C of incubated overnights of bacterium solution, the big extraction reagent kit of endotoxin-free plasmid extracts plasmid, and adopts
Plasmid concentration is determined with micro ultraviolet specrophotometer.
Further, picking monoclonal carries out the checking of 10ul systems bacterium solution PCR after the conversion coated plate, specifically includes：Picking
Monoclonal 0.5ul bacterium solutions, 0.5ul YSY checking forward primers, 0.5ul R-Oligo, 5ul Mastermix, 3.5ul
MilliQ；PCR reaction conditions are：1)：95 DEG C of denaturations 2mim；2)：94 DEG C of denaturation 30s；3)：56 DEG C of annealing 30s；4)：72℃
Extend 30s；Step 2) to step 4) 35 circulations of operation；5)：72 DEG C re-extend 10min；6)：4 DEG C of preservations.
Further, the primer includes：
F-MC1R-gRNA-L1：CACCGAAGGAGAGGGAGGACACGA；
R-MC1R-gRNA-L1：AAACTCGTGTCCTCCCTCTCCTTCC；
F-MC1R-gRNA-R1：CACCGCTTCCTGGGGGTCATCGCCG；
R-MC1R-gRNA-R1：AAACCGGCGATGACCCCCAGGAAG.
Further, the plasmid concentration is：
PYSY-CMV-Cas9n-U6-MC1R-gRNA-L2-SV40-Neo plasmids：4ug concentration:190ng/ul；
PYSY-CMV-Cas9n-U6-MC1R-gRNA-R2-EF1a-eGFP plasmids：4ug concentration:184ng/ul.
The MC1R genophores and its construction method that the present invention is provided adopts CRISPR/cas9 systems, designs MC1R genes
SgRNA fragments, chemical synthesis sgRNA nucleotide sequences build the plasmid PYSY- for expressing sgRNA and Cas9 D10A simultaneously
SgRNA, connects and converts to bacillus coli DH 5 alpha competent cell, and finally transformant is verified；Digestion and sequencing identification
Prove that MC1R gene knockout carriers build correct.The carrier that the present invention is built using CRISPR/Cas9, can orient knockout chicken
MC1R genes, thus the phases such as chicken MC1R Gene Deletion cell lines, production MC1R gene delection transgenic chickens can be obtained for follow-up
Close research to lay the foundation.The present invention, by building MC1R gene knockout carriers, is subsequently to prepare MC1R genes using the carrier to lack
Cell line is lost, further to study MC1R gene functions basis is provided.
The present invention adopts CRISPR/Cas9 system constructing MC1R gene knockout carriers, and method simple and fast only need to be for being somebody's turn to do
A sgRNA for being about 20bp or so is designed in gene knockout site, then the Cas9 genes of connection universal, and adopts ZFNs
Or TALENs carries out gene knockout, each gene loci editor is required for design and assemble two nucleases, constructing technology is difficult
Spend larger, the component composition time longer.Therefore, with the gene Knockout such as traditional ZFNs, TALENs comparatively, using
CRISPR/Cas9 builds gene knockout carrier more simple and fast, is easy to further genralrlization and is applied to subsequent experimental.
With ZFN and TALEN, both artificial nucleases are compared, and the Cas9 in CRISPR/Cas9 systems is used as nickase, tool
There is single-stranded cleavage activity, single-stranded nick can be manufactured in ad-hoc location, non-homologous end joining so will not be caused substantially, so as to
Efficiently the fixed point of mediate foreign gene is knocked in, or carries out point mutation to genome, greatly reduces non-homologous end joining institute
The risk brought.
The present invention forms two incision using the CRISPR-Cas9 systems that RNA is oriented to, before not affecting to target cutting efficiency
Put and greatly reduce effect of missing the target, improve gene knockout efficiency, relative to traditional single otch knockout carrier, it knocks out efficiency
More than 20% can be improved.
Description of the drawings
Fig. 1 is the construction method flow chart of MC1R genophores provided in an embodiment of the present invention.
Fig. 2 is PCR checkings clone's electrophoresis picture example schematic diagram provided in an embodiment of the present invention；
In figure：Marker：Trans 2K Plus DNA Marker, are followed successively by from top to bottom 100bp, 250bp, 500bo,
750bp, 1000bp, 3000bp, 5000bp；2:Negative control；3-6:MC1R-gRNA-Rg1 is cloned.
Fig. 3 is PCR checkings clone's electrophoresis picture example schematic diagram provided in an embodiment of the present invention；
In figure：Marker：Trans 2K Plus DNA Marker, are followed successively by from top to bottom 100bp, 250bp, 500bo,
750bp, 1000bp, 3000bp, 5000bp；2:Negative control；3-6:MC1R-gRNA-Lg1 is cloned.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that specific embodiment described herein is not used to only to explain the present invention
Limit the present invention.
The application principle of the present invention is explained in detail below in conjunction with the accompanying drawings.
The MC1R genophores of the embodiment of the present invention include MC1R-neo and MC1R-GFP；The MC1R-neo nucleotides sequences
It is classified as SEQ ID NO：1；The MC1R-GFP nucleotides sequences are classified as SEQ ID NO：2.
SEQ ID NO：1 is：
TGGCTTTATATCTTGTGGAAAGGACGAAACACCGAAGGAGAGGGAGGACACGAGTTTTAGAGCTAGAA；
SEQ ID NO：2 are：
TGGCTTTATATCTTGTGGAAAGGACGAAACACCGCTTCCTGGGGGTCATCGCCGGTTTTAGAGCTAGA。
As shown in figure 1, the construction method of the MC1R genophores of the embodiment of the present invention is comprised the following steps：
S101：Primer annealing：1ul F-Oligo (100uM), 1ul R-Oligo (100uM), 8ul YSY oligo anneal
Buffer solution, above solution is mixed in PCR pipe, and gradually 22 DEG C are down to from 95 DEG C with 1.5 DEG C per minute in PCR instrument；
S102：Connection：0.5ul annealed products, 1ul YSY linearize three-in-one CRISPR/Cas9n plasmids, and 1ul T4 connect
Meet enzyme, 2ul 5*T4Buffer, 5.5ul Milli Q；
S103：Conversion：Escherichia coli DH5a competent cell (pfu >=10 are used after room temperature 15min8) converted；
S104：Transformant is verified：Picking monoclonal carries out the checking of 10ul systems bacterium solution PCR after conversion coated plate；
0.5ul bacterium solutions, 0.5ul YSY checking forward primers, 0.5ul R-Oligo, 5ul Mastermix, 3.5ul
MilliQ；PCR reaction conditions are：1)：95 DEG C of denaturations 2mim；2)：94 DEG C of denaturation 30s；3)：56 DEG C of annealing 30s；4)：72℃
Extend 30s；Step 2) to step 4) 35 circulations of operation；5)：72 DEG C re-extend 10min；6)：4 DEG C of preservations；
S105:It is sequenced Jing after PCR Preliminary Identifications；
S106：Plasmid extraction：37 DEG C of incubated overnights of bacterium solution, the big extraction reagent kit of endotoxin-free plasmid extracts plasmid, and adopts
Micro ultraviolet specrophotometer determines plasmid concentration.
The application principle of the present invention is further described with reference to experiment.
1 material and method
1.1 materials
Trans2K Plus DNA Marker are purchased from Quan Shijin Bioisystech Co., Ltd, linearize three-in-one CRISPR/
Cas9n plasmids are purchased from Nanjing Yao and Shun Yu company, and escherichia coli DH5a competent cell, T4 ligases, endotoxin-free plasmid are carried greatly
Kit is purchased from Tiangeng biochemistry Co., Ltd.
1.2 methods
1.2.1 sgRNA target spots determine and design of primers
According to chicken MC1R gene (GenBank:KF379749.1), find CDS sequences and analyze its structure, according to the gene
Structure determination knocks out site, selects exon sequence to be input in software, obtains gRNA sequences.And 2 generated according to design
SgRNA, designs and synthesizes corresponding 2 couples of primer Oligo as follows：
SEQ ID NO：3 F-MC1R-gRNA-L1：CACCGAAGGAGAGGGAGGACACGA；
SEQ ID NO：4 R-MC1R-gRNA-L1：AAACTCGTGTCCTCCCTCTCCTTCC；
SEQ ID NO：5 F-MC1R-gRNA-R1：CACCGCTTCCTGGGGGTCATCGCCG；
SEQ ID NO：6 R-MC1R-gRNA-R1：AAACCGGCGATGACCCCCAGGAAG；
1.2.2 the structure of CRISPR/Cas9 gene knockout carriers
Primer annealing：1ul F-Oligo (100uM), 1ul R-Oligo (100uM), 8ul YSY oligo annealing buffers
Liquid, above solution is mixed in PCR pipe, and gradually 22 DEG C are down to from 95 DEG C with 1.5 DEG C per minute in PCR instrument.
Connection：0.5ul annealed products, the three-in-one CRISPR/Cas9n plasmids of 1ul YSY linearisations, 1ul T4 ligases,
2ul 5*T4 Buffer, 5.5ul Milli Q.
Conversion：Escherichia coli DH5a competent cell (pfu >=10 are used after room temperature 15min8) converted.
Transformant is verified：Picking monoclonal carries out the checking of 10ul systems bacterium solution PCR after conversion coated plate.
0.5ul bacterium solutions, 0.5ul YSY checking forward primers, 0.5ul R-Oligo, 5ul Mastermix, 3.5ul
MilliQPCR reaction conditions：Seg1：95 DEG C of denaturations 2mim；Seg2：94 DEG C of denaturation 30s；Seg3：56 DEG C of annealing 30s；Seg4：
72 DEG C of extension 30s；Seg2to Seg4 run 35 circulations；Seg5：72 DEG C re-extend 10min；Seg6：4 DEG C of preservations.
Jing PCR are initially identified as positive colony and deliver to Nanjing Genscript Biotechnology Co., Ltd. being sequenced.
1.2.3 plasmid extraction
37 DEG C of incubated overnights of bacterium solution, the big extraction reagent kit of endotoxin-free plasmid extracts plasmid, and using micro ultraviolet spectrometry light
Degree meter determines plasmid concentration.
2. result
2.1 sgRNA are designed
SgRNA identifications position is located on the corresponding positions of 7tm_1 (after 60aa or after total 120aa)
The sgRNA transcript folder RNA sequences being expected under U6 promoters drivens are respectively：
SEQ ID NO：7 are：MC1R-gRNA-Lg1
GAAGGAGAGGGAGGACACAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA
AAGUGGCACCGAGUCGGUGCUUUUUU；
SEQ ID NO：8 are：MC1R-gRNA-Rg1
GCUUCCUGGGGGUCAUCGCCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA
AAGUGGCACCGAGUCGGUGCUUUUUU。
2.2 PCR checking positive colony electrophoresis picture is as shown in Figures 2 and 3.
2.3 censorship sequencing results
To before verify that correct positive colony bacterium solution delivers to the sequencing of Jin Sirui Bioisystech Co., Ltd, partial order will be sequenced
Row comparison result is specific as follows：
2.3.1 pYSY-CMV-Cas9n-U6-MC1R-gRNA-L2-SV40-Neo
By contrast, its homology reaches 100%.
2.3.2 pYSY-CMV-Cas9n-U6-MC1R-gRNA-R2-EF1a-Egfp
Its homology reaches 100%.Therefore, by above sequence alignment, it may be determined that target plasmid is successfully constructed.
The knockout plasmid pair of 2.4 chicken MC1R genes is extracted and concentration mensuration：
PYSY-CMV-Cas9n-U6-MC1R-gRNA-L2-SV40-Neo plasmids：4ug concentration:190ng/ul.
PYSY-CMV-Cas9n-U6-MC1R-gRNA-R2-EF1a-eGFP plasmids：4ug concentration:184ng/ul.
Presently preferred embodiments of the present invention is the foregoing is only, not to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
<110>University of Anhui
<120>A kind of construction method of MC1R genophores
<160> 8
<210> 1
<211> 69
<212>DNA
<213>Chicken MC1R
<400>Nucleotide sequence
TGGCTTTATATCTTGTGGAAAGGACGAAACACCGAAGGAGAGGGAGGACACGAGTTTTAGAGCTAGAA
<210> 2
<211> 69
<212>DNA
<213>Chicken MC1R
<400>Nucleotide sequence
TGGCTTTATATCTTGTGGAAAGGACGAAACACCGCTTCCTGGGGGTCATCGCCGGTTTTAGAGCTAGA
<210> 3
<211> 24
<212>RNA
<213> F-MC1R-gRNA-L1
<400>Nucleotide sequence
CACCGAAGGAGAGGGAGGACACGA
<210> 4
<211> 25
<212>RNA
<213> R-MC1R-gRNA-L1
<400>Nucleotide sequence
AAACTCGTGTCCTCCCTCTCCTTCC
<210> 5
<211> 25
<212>RNA
<213> F-MC1R-gRNA-R1
<400>Nucleotide sequence
CACCGCTTCCTGGGGGTCATCGCCG
<210>6
<211> 24
<212>RNA
<213> R-MC1R-gRNA-R1
<400>Nucleotide sequence
AAACCGGCGATGACCCCCAGGAAG
<210>7
<211> 103
<212>RNA
<213>Chicken MC1R
<400>Nucleotide sequence
GAAGGAGAGGGAGGACACAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
GAAAAAGUGGCACCGAGUCGGUGCUUUUUU
<210>8
<211> 103
<212>RNA
<213>Chicken MC1R
<400>Nucleotide sequence
GCUUCCUGGGGGUCAUCGCCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
GAAAAAGUGGCACCGAGUCGGUGCUUUUUU
Claims (5)
1. a kind of MC1R genophores, it is characterised in that the MC1R genophores include MC1R-neo and MC1R-GFP；
The MC1R-neo nucleotides sequences are classified as SEQ ID NO：1；The MC1R-GFP nucleotides sequences are classified as SEQ ID NO：2.
2. a kind of construction method of MC1R genophores as claimed in claim 1, it is characterised in that the construction method include with
Lower step：
Step one, primer annealing：1ul F-Oligo (100uM), 1ul R-Oligo (100uM), 8ul YSY oligo annealing is slow
Liquid is rushed, above solution is mixed in PCR pipe, gradually 22 DEG C are down to from 95 DEG C with 1.5 DEG C per minute in PCR instrument；
Step 2, connection：0.5ul annealed products, 1ul YSY linearize three-in-one CRISPR/Cas9n plasmids, 1ul T4 connections
Enzyme, 2ul 5*T4Buffer, 5.5ul Milli Q；
Step 3, conversion：Pfu >=10 are used after room temperature 15min8Escherichia coli DH5a competent cell converted；
Step 4, transformant checking：Picking monoclonal carries out the checking of 10ul systems bacterium solution PCR after conversion coated plate；
Step 5, is sequenced Jing after PCR Preliminary Identifications；
Step 6, plasmid extraction：37 DEG C of incubated overnights of bacterium solution, the big extraction reagent kit of endotoxin-free plasmid extracts plasmid, and using micro-
Amount ultraviolet specrophotometer determines plasmid concentration.
3. the construction method of MC1R genophores as claimed in claim 2, it is characterised in that picking list after the conversion coated plate
Clone carries out the checking of 10ul systems bacterium solution PCR, specifically includes：Picking monoclonal 0.5ul bacterium solutions, 0.5ul YSY checkings forward direction is drawn
Thing, 0.5ul R-Oligo, 5ul Mastermix, 3.5ul MilliQ；PCR reaction conditions are：1)：95 DEG C of denaturations 2mim；
2)：94 DEG C of denaturation 30s；3)：56 DEG C of annealing 30s；4)：72 DEG C of extension 30s；Step 2) to step 4) 35 circulations of operation；5)：72
DEG C re-extend 10min；6)：4 DEG C of preservations.
4. the construction method of MC1R genophores as claimed in claim 2, it is characterised in that the primer includes：
F-MC1R-gRNA-L1：CACCGAAGGAGAGGGAGGACACGA；
R-MC1R-gRNA-L1：AAACTCGTGTCCTCCCTCTCCTTCC；
F-MC1R-gRNA-R1：CACCGCTTCCTGGGGGTCATCGCCG；
R-MC1R-gRNA-R1：AAACCGGCGATGACCCCCAGGAAG.
5. the construction method of MC1R genophores as claimed in claim 2, it is characterised in that the plasmid concentration is：
PYSY-CMV-Cas9n-U6-MC1R-gRNA-L2-SV40-Neo plasmids：4ug concentration:190ng/ul；
PYSY-CMV-Cas9n-U6-MC1R-gRNA-R2-EF1a-eGFP plasmids：4ug concentration:184ng/ul.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108559730A (en) * | 2018-01-12 | 2018-09-21 | 中国人民解放军第四军医大学 | Hutat2 is built using CRISPR/Cas9 technologies:The experimental method of Fc gene knock-in monocytes |
| CN108559730B (en) * | 2018-01-12 | 2021-09-24 | 中国人民解放军第四军医大学 | Experimental method for constructing Hutat2 Fc gene knock-in monocyte by CRISPR/Cas9 technology |
| CN108424929A (en) * | 2018-01-23 | 2018-08-21 | 中山大学 | The Cas9/sgRNA coexpression vectors and its construction method of pig MC1R genes and application |
| CN110372787A (en) * | 2019-07-24 | 2019-10-25 | 上海交通大学 | 1 albumen of Chinese ring-necked pheasant melanocortin receptor and its encoding gene and application |
| CN111235154A (en) * | 2020-04-01 | 2020-06-05 | 陕西慧康生物科技有限责任公司 | sgRNA for targeted knockout of human MC1R gene and cell strain constructed by sgRNA |
| CN111235153A (en) * | 2020-04-01 | 2020-06-05 | 陕西慧康生物科技有限责任公司 | sgRNA for targeted knockout of human MC1R gene and cell strain constructed by sgRNA |
| CN111235153B (en) * | 2020-04-01 | 2023-05-26 | 陕西慧康生物科技有限责任公司 | sgRNA for targeted knockout of human MC1R gene and cell strain constructed by same |
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