CN106399371B - A method of building gene substitution mice study wild type and the non-flesh myoglobulin heavy chain II function of mutant - Google Patents
A method of building gene substitution mice study wild type and the non-flesh myoglobulin heavy chain II function of mutant Download PDFInfo
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Abstract
The present invention provides a kind of methods for constructing gene substitution mice study wild type and the non-flesh myoglobulin heavy chain II function of mutant.For the mpNTKV-LoxP carrier that the present invention is transformed more suitable for building gene targeting carrier especially gene substitution purpose, Pac I and the Swa I being newly added are the restriction enzymes for identifying eight bases, are conducive to the insertion of larger dna segment.MYH9 gene substitution (i.e. endogenous MYH9 gene is substituted by the non-flesh myoglobulin heavy chain II gene of the outer source code) targeting vector of building is transduceed by electricity in ES cells, by drug screening, ES cell monoclonal is selected, Southern Blot or PCR identification, the positive monoclonal cell rate that homologous recombination occurs is up to 90% or more, is easily obtained required cell line.MYH9 gene substitution allophenic mice is obtained using positive ES cells microinjection to Mouse Blastocysts, and the allophenic mice can mate passage, to obtain MYH9 gene substitution heterozygote and homozygote offspring.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of building gene substitution mice study wild type and mutant
The method and its application of non-flesh myoglobulin heavy chain II function are demonstrated
Background technique
Gene knockout is a kind of novel molecular biology skill for applying DNA homologous recombination principle to grow up the end of the eighties
Art, i.e., the technology for making the specific gene of body inactivate or lack by certain approach.The technology development experience three main
Process: i.e. the separation of ES at beginning of the eighties cell and in vitro culture are that gene knockout has established technical foundation；It confirms within 1985 to feed for the first time
The gene knockout that exists for of homologous recombination phnomena has established theoretical basis in newborn zooblast；Completely it is based on ES cell within 1987
The foundation of gene knock-out mice model indicate the maturation of the technology.The conditionity or inductivity to grow up on this basis
Gene knockout system make to gene modification over time and space definitely, effect it is more perfect, thus research can
It can lead to some important genes of embryonic death, and have any actual knowledge of some gene in specific stage of development or the function of specific organization
Important function has been played in energy.Dependence Zinc finger nuclease (ZFN), the transcription factor sample effector nuclease occurred in recent years
(TALEN) and the short palindrome in Regularity interval repeats and its genome editing technique of related protein system (CRISPR/Cas9)
Further push the development and application of gene knockout.It should be pointed out that these new technologies there are still building it is complicated or
The problems such as flexibility is poor or undershooting-effect, is also in the incomplete stage of ripeness.Therefore, it is based on ES cell and benefit even to this day
Gene knockout, which is carried out, with DNA homologous recombination is still most reliable and most common user in building Gene Knock-Out Animal Model model
Method.
There are numerous genes or protein family in organism, their structure and functions are similar.The non-flesh flesh ball egg of II type
White (Nonmuscle Myosin II, NM II) is a member of myosin superfamily, point being bonded in cell with actin
Sub- motor.All myosin II have similar structure, i.e., by two heavy chains (171-244kDa), two adjusting light chains and two
Six aggressiveness of required light chain (16-23kDa) composition of item.The amino terminal that two pairs of light chains are incorporated into a pair of of heavy chain forms ballhead
Portion has atpase activity and actin binding site；The carboxyl terminal of heavy chain passes through the rod-shaped tail portion of coiled-type spiralization.It feeds
NM II point is three kinds of hypotypes in newborn animal, this is the difference by heavy chain and determines.Three genes (MYH9, MYH10, MYH14)
Be separately encoded NM heavy chain II (Nonmuscle myosin heavy chain II, NMHC II) NMHCIIA, NMHCIIB and
NMHCIIC.Six dimeric complexes that a pair of identical heavy chain is formed in conjunction with two pairs of light chains are respectively NM IIA, NM IIB, NM
IIC.NM II other than providing power for cell movement, also participation the various physiological activities of cell, as cell migration and stick,
Cytokinesis etc..MYH9 and MYH10 gene is inactivated respectively in mouse using gene Knockout, discloses these NM II eggs
It is white required for mouse early development.Simultaneously as the embryonic lethality of MYH9 and MYH10 knock out mice, limit into
One step understands the function of these albumen in vivo；More than one NM II hypotype is expressed in most cells, these are different sub-
Specific function is still unclear in vivo by type NM II；There is centainly special in expression of the different subtype NM II in cell and tissue
Property, does this cause the difference of their functions? in addition, the single point mutation of MYH9 gene causes one kind to be referred to as MYH9 correlation
Disease is currently known and the MYH9 point mutation of this kind of disease is caused to be as high as more than 40, spreads over NMHC IIA albumen everywhere,
If analyzing the reason of these point mutation cause disease one by one will need to produce more than 40 corresponding MYH9 gene origin mutant mice,
It works undoubtedly extremely numerous huge.Using gene substitution strategy, it will effectively inquire into, solve and answer the above problem.
Summary of the invention
The present invention is directed to overcome the shortcomings of existing research strategy and technical method, a kind of building gene substitution mouse is provided and is ground
The method for studying carefully wild type and the non-flesh myoglobulin heavy chain II function of mutant.
In order to achieve the above object, technical solution provided by the invention are as follows:
The method of framework gene substitution the mice study wild type and the non-flesh myoglobulin heavy chain II function of mutant
Include the following steps:
(1) mpNTKV-LoxP carrier is constructed, the mpNTKV-LoxP carrier sequence is as shown in SEQ ID NO.4；
(2) targeting vector of targeting MYH9 gene is constructed on the basis of mpNTKV-LoxP carrier: will be outside mouse MYH9 gene
Aobvious 2 upstream 4.0kb of son and downstream 1.7kkb DNA sequence dna, which are inserted into respectively in mpNTKV-LoxP carrier, obtains targeting MYH9 gene
Targeting vector is named as mpNTKV-LoxP MAL-MAR targeting vector, the mpNTKV-LoxP MAL-MAR targeting vector sequence
Column are as shown in SEQ ID NO.25；In addition, the homology arm sequence of the mpNTKV-LoxP MAL-MAR targeting vector is by mouse
2 upstream and downstream of MYH9 gene extron about 6kb DNA sequence dna composition, as shown in SEQ ID NO.24；
(3) by the cDNA of the non-flesh myoglobulin heavy chain II of the coding fusion fluorescin of the quasi- endogenous MYH9 gene of substitution
(i.e. mCherry-NMHC IIA, GFP-NMHC IIB, GFP-NHMC IIA R702C) insertion targets MYH9 gene targeting respectively
To obtain three different MYH9 gene substitution targeting vectors in carrier, these targeting vectors are respectively designated as in the present invention
Targeting vector 1,2,3, as shown in Figure 2；Their sequence is respectively such as SEQ ID NO.26, SEQ ID NO.27, SEQ ID
Shown in NO.28；
(4) the MYH9 gene substitution series targeting vector of building is passed through electricity respectively to transduce in ES cells, through medicine
Object screens to obtain cell monoclonal, through Southern Blot or PCR identification of cell monoclonal, and then obtains MYH9 gene substitution
ES cell line, the MYH9 gene substitution ES cell line is for generating MYH9 gene substitution mouse.
Wherein, drug screening described in step (4) is through G418 and Ganciclovir drug screening.
The invention will be further described below:
The main purpose of the present invention is to provide one kind based on ES cell construction gene substitution mice study wild type and to dash forward
The method of variant NMHC II function.It specifically includes that the mpNTKV-LoxP carrier of 1) transformation, has more for DNA fragmentation
The restriction enzyme site of insertion, and the addition in the two sides the resistant gene Neomyocin site LoxP, so that the removal of resistant gene has
It may；2) DNA sequence dna of the left and right homology arm of the targeting vector of building targeting MYH9 gene, is constructed using these DNA sequence dnas
Targeting vector carries out MYH9 gene substitution its homologous recombination efficiency of practicing shooting in ES cells and is up to 90% or more；3) screening with
Identify the Southern Blot probe and PCR primer of positive ES cells monoclonal and murine genes type.
Firstly, the present invention carries out first using sequence primer pair as shown in SEQ ID NO.1 and SEQ ID NO.2
Secondary PCR；On this basis, second of PCR is carried out using sequence primer pair as shown in SEQ ID NO.1 and SEQ ID NO.3.
Original vector pNTKV1905 and second of PCR product pass through digestion, the recycling of DNA glue, connection, conversion, Plasmid DNA preparation respectively, survey
Sequence and etc., as a result confirm vector modification success, new support is named as mpNTKV-LoxP, and map is as shown in Figure 1, complete sequence
Such as SEQ ID NO.4.In addition, two pairs of primers of design facilitate the DNA fragmentation sequencing of insertion two multiple cloning sites of new support, primer
Position as shown in Figure 1, primer sequence as shown in SEQ ID NO.5,6,7,8.
Secondly, according to sequence shown in SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12
Information makees template using the BAC DNA of the gene of MYH9 containing mouse and carries out the left and right homology arm of PCR amplification respectively, then will be left and right same
Source arm is inserted into mpNTKV-LoxP plasmid, and the targeting vector for obtaining targeting MYH9 gene is as shown in Figure 2.Right side homology arm sequence
As shown in SEQ ID NO.13, left side homology arm sequence is as shown in SEQ ID NO.14.On the basis of constructing above-mentioned targeting vector,
Quasi- wild type NMHC IIA or the cDNA coding for implementing substitution cDNA coding fusion red fluorescent protein (mCherry) is melted
Close NMHC IIB or cDNA coding fusion the green fluorescent protein R702C mutant NMHC IIA of green fluorescent protein (GFP)
And between SV40polyA termination sequence insertion left side homology arm and resistant gene Neomyocin, thus obtain three it is different
MYH9 gene substitution targeting vector, as shown in Figure 2.
The targeting vector of linearisation is directed respectively into ES cells by electric shifting method, is added after 24 hours by third
G418 and Ganciclovir is carried out drug screening one week, then selects ES cell monoclonal, and carry out cell amplification, extracts base
Because group DNA is for screening and identifying.
4th, the complete genome DNA extracted from ES cells is subjected to digestion, the separation of DNA glue, transferring film, and through putting
The left and right DNA probe hybridization of injectivity isotope label, the Southern Blot process such as wash film and radioactive automatic developing, thus really
Determine whether ES cell monoclonal is the homologous recombination positive.Left and right side probe location is as shown in Figure 2；Probe sequence on the left of PCR amplification
Such as SEQ ID NO.15 and SEQ ID NO.16；Probe sequence such as SEQ ID NO.17 and SEQ ID NO.18 on the right side of PCR amplification.
Positive cell monoclonal can also be determined by PCR using SEQ ID NO.19 and SEQ ID NO.20.In addition, positive cell
The genotype of monoclonal and gene substitution mouse is also using SEQ ID NO.21, SEQ ID NO.22 and SEQ ID NO.23
Primer is determined by PCR.
Compared with the prior art and method, present invention has an advantage that
1) mpNTKV-LoxP carrier is more convenient the building for being inserted into be conducive to gene substitution targeting vector of DNA fragmentation,
It is up to 13kb by the DNA fragmentation length of three steps clone's insertion, wherein the cDNA segment substituted reaches 7kb, can satisfy completely big
Most gene insertions require, and the site LoxP that the two sides resistant gene Neomyocin are furthermore added can according to need, and utilize Cre
Implementation cuts off it.
2) target is constructed using first upstream ATG 4.0kb DNA sequence dna of mouse MYH9 gene and downstream 1.7kb DNA sequence dna
It is encoded to the left and right homology arm of carrier, then by the cDNA encoding wild type for implementing substitution or mutant NMHC IIA or cDNA
NMHC IIB is placed between left side homology arm and resistant gene Neomyocin, obtains gene substitution targeting vector.So exquisite peace
Row is so that endogenous MYH9 gene is destroyed and expresses the gene knocked in.Most of all, being realized using these targeting vectors homologous
The efficiency of recombination is up to 90% or more, and positive ES cells monoclonal is easily obtained.
3) Southern Blot probe, PCR primer provided by make the sieve of positive cell monoclonal, murine genes type
It selects and identifies and is more flexible and convenient.
The present invention constructs mpNTKV-LoxP carrier, is transformed through PCR, digestion and connection by pNTKV1905.Newly
One multiple cloning sites of carrier joined Pac I, Nhe I, Swa I restriction enzyme site, and another multiple cloning sites joined Pme I enzyme
Enzyme site, and the two sides resistant gene Neomyocin have been separately added into the site LoxP, so that and the carrier is more advantageous to DNA
Segment relies on the insertion of restriction enzyme site and the subsequent removal of resistant gene.
Constructing MYH9 gene, (it encodes non-flesh myoglobulin heavy chain IIA, Nonmuscle Myosin Heavy
Chain IIA, NMHC IIA) substitution targeting vector, which includes MYH9 gene the second exon upstream sequence of 4.0kb
Left side homology arm, cDNA coding human wild type or the expression cassette of mutant NMHC II, positive resistant gene Neomyocin,
The right side homology arm and negative resistance TK gene of MYH9 gene the second exon downstream sequence of 1.7kb.
The gene substitution targeting vector linearized through Not I is imported in ES cells using electric shifting method, through positive and negative
After labeled drug screening, ES cell monoclonal is separated, Southern blot or PCR identification confirm that the gene of homologous recombination occurs
Target practice efficiency is more than 90%.Multiple (>=3) endogenous MYH9 gene is obtained by human wild type or mutant NMHC based on this method
The ES cell line of II substitution and corresponding mouse species.Therefore, carrier provided by the present invention, DNA sequence dna, ES cell monoclonal
A kind of method of screening and identification, for research NMHC II gene family is normal or the function of mutain provides new method.
In short, the mpNTKV-LoxP carrier that the present invention is transformed is replaced more suitable for building gene targeting carrier especially gene
For purpose, Pac I and the Swa I being newly added are the restriction enzymes for identifying eight bases, are conducive to larger dna segment
Insertion.The MYH9 gene substitution targeting vector of building is transduceed by electricity in ES cells, by drug screening, ES cell list
Clone selects, and Southern Blot or PCR identification, the positive monoclonal cell rate that homologous recombination occurs are up to 90% or more, easily
The cell line needed for obtaining.MY9 gene substitution allophenic mice is obtained using positive ES cells microinjection to Mouse Blastocysts, and
And the allophenic mice can mate passage, to obtain MYH9 gene substitution heterozygote and homozygote offspring.
Detailed description of the invention
Fig. 1: mpNTKV-LoxP Vector map and sequencing primer position；
The building schematic diagram of Fig. 2: MYH9 gene substitution targeting vector, Southern blot identify restriction enzyme used
Each mutation allele behind the positions such as enzyme, probe and PCR identification primer and gene substitution；
Fig. 3: Southern blot screening mouse embryo stem cell monoclonal is carried out using right side probe to be mutated
AmChAllele, wild-type allele (A+) stripe size is expected as 9.7kb, mutant allele (AmCh) it is expected band
Size is 6.0kb；
Fig. 4: the A of P4+P5 primer pair gene substitution is utilizedmChAllele mouse embryo stem cell monoclonal carries out PCR and tests
Card, wild-type allele (A+) without band, mutant allele includes AmChAnd Ab*And AgfpR702CThen it is amplifiable go out about
2.0kb band, ES cell monoclonal serial number are corresponding with the serial number in Southern Blot analysis；
Fig. 5: PCR analysis, wild type (A are carried out using P1+P2+P3 primer pair gene substitution mice progeny genotype+) etc.
Position gene magnification goes out 420bp band, mutant allele (AmCh、Ab*And AgfpR702C) amplify 800bp band；
Fig. 6: Laser Scanning Confocal Microscope discloses substitution gene mCherry-NMHC IIA and GFP-NMHC IIB in A+/AmChAnd A+/Ab*It is correctly expressed in fibroblast made of mouse embryo stem cell differentiation；
Fig. 7: R702C point mutation NMHC IIA substitutes hybrid mice (A+/AgfpR702C) show and mankind's gene loci
It is mutated consistent clinical phenotypes.With wild-type mice (A+/A+) compare, A+/AgfpR702CMouse shows Giant platelet (above)
With glomerulosclerosis (following figure).
Specific embodiment
Embodiment 1: the building of plasmid mpNTKV-LoxP
To make two multiple cloning sites of original vector pNTKV1905 (purchased from Stratagene) possess more restriction enzyme sites,
And the site LoxP is added in the two sides resistant gene Neomycin, makees template with pNTKV1905 and utilizes forward primer mpN-LoxP-
F5’-AGGAAGCTTGTTTAAACATAACTTCGTATAATGTATGCTATACGAAGTTATTCGAGGGGCGCGCCCCCAGCT
GGTTCTTTCCGCC (SEQ ID NO.1) and reverse primer mpN-LoxP-R15 '-AGCCATATGATTTAAATGAATTCATA
ACTTCGTATAGCATACATTATACGAAGTTATCATATGTGATCCCGGCGCGCCCTACCGGGTAGGGGAGGCGCTT
(SEQ ID NO.2) and Platinum Taq DNA Polymerase High Fidelity (Invitrogen) make PCR amplification
(94 DEG C of 2min, 94 DEG C of 30s, 60 DEG C of 30s, 68 DEG C of 2min, 30cycles, 68 DEG C of 10min), PCR product is through PCR
Clean-up kit (being purchased from Qiagen) purifying.Then template is made with the purified product and utilizes forward primer mpN-LoxP-F 5 '-
AGGAAGCTTGTTTAAACATAACTTCGTATAATGTATGCTATACGAAGTTATTCGAGGGGCGCGCCCCCAGCTGGTT
CTTTCCGCC and reverse primer mpN-LoxP-R25 '-CGCGTCGACTCTAGGATCGATTCTAGCCGCGGCCCCCCGGGTG
TAGTTAATTAAGCTAGCCATATGATTTAAATGAATTC(SEQ ID NO.3) and Platinum Taq DNA
Second of PCR amplification of Polymerase High Fidelity work (94 DEG C of 2min, 94 DEG C of 30s, 60 DEG C of 30s, 68 DEG C
2min, 30cycles, 68 DEG C of 10min), underlined sequences are restriction enzyme site and two reverse primer laps, PCR product warp
PCR clean-up kit purifying.Second of PCR purified product and carrier pNTKV1905 are respectively through Sal I and Hind III (purchase
From after Fermantas) double digestion, is separated using 1.0% agarose gel electrophoresis, utilize DNA Gel Extraction kit
(being purchased from Qiagen) recycling about 3.8kb carrier and 1.8kb PCR band, even using T4DNA Ligase (being purchased from Fermantas)
It connects, converts bacillus coli DH 5 alpha, Plasmid DNA is extracted in picking single bacterium colony culture, is surveyed through digestion Preliminary Identification Hou Song commercial company
Sequence.Sequencing result shows that carrier is successfully modified and be named as mpNTKV-LoxP, and map is as shown in Figure 1 and complete sequence institute SEQ
Shown in ID NO.4.For the DNA sequence dna of confirmation insertion two multiple cloning sites of new support, the ad hoc forward and reverse sequencing of two pairs of meter is drawn
Object, sequencing primer position as shown in Figure 1, sequencing primer sequence such as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7,
Shown in SEQ ID NO.8.
Embodiment 2: the building of the left and right homology arm of targeting vector of targeting MYH9 gene
From genome.ucsc.edu retrieved web and downloading obtains mouse MYH9 genome sequence, in conjunction with mouse
MYH9mRNA (GenbankNo:NM_022410) sequence determines each exon and introne position and sequence, especially contains first
The exon of a ATG.According to each 6kb genome sequence in the second exon of MYH9 (exon locating for first ATG) upstream and downstream
Column choose optimal sequence using Primer3 software design 4kb or so is long-armed and 2kb or so galianconism PCR primer.Referring to David
2005 document report of J.Adams, acquisition include the BAC Clone No:bMQ-369E5 of entire MYH9 gene, and from
Chieldren ' hospotal Oakland Research Institute purchase obtains, and BAC DNA utilizes QIAGEN
Plasmid Midi Kit (Qiagen) prepares spare.Forward primer is utilized by template of bMQ-369E5BAC DNA
MYH9Exon2R-F(Kpn I)5’-GGGGTACCGGCTCAGCAGGCTGCAGACAAGTACCTC (SEQ ID NO.9) and reversed
Primer MYH9Exon2R-R (BamH I) 5 '-CGGGATCCCAGCGGGGTAGGAAGCACGATG (SEQ ID NO.10) and
Platinum Taq DNA Polymerase High Fidelity makees on the right side of PCR amplification about 1.7kb (94 DEG C of homology arm
2min, 94 DEG C of 30s, 60 DEG C of 30s, 68 DEG C of 2min, 30cycles, 68 DEG C of 10min), underlined sequences are restriction enzyme site.
PCR product carries out double digestion, and empty carrier mpNTKV- using Kpn I and BamH I after purification through PCR clean-up kit
LoxP carries out double digestion using Kpn I and Bgl II and cuts out purpose band after agarose gel electrophoresis separates and recycle, so
Carrier is connect with target fragment afterwards, is then converted to bacillus coli DH 5 alpha, picking single bacterium colony culture, Plasmid DNA is extracted, through enzyme
Cut the sequencing of Preliminary Identification Hou Song commercial company.Sequencing result display right side homology arm is inserted into successfully and is named as mpNTKV-LoxP
MAR, right side homology arm sequence is as shown in SEQ ID NO.13.Forward primer is utilized by template of bMQ-369E5BAC DNA
MYH9Exon2L-F(Sal I)5’-ACGCGTCGACGAAGTGAAGCTCCTGGCTTTG (SEQ ID NO.11) and reverse primer
MYH9Exon2L-R(Sac II)5’-TCCCCGCGGGTGACTTGCGGCCAGGACCTAAG (SEQ ID NO.12) and
Platinum Taq DNA Polymerase High Fidelity makees on the left of PCR amplification about 4.0kb (94 DEG C of homology arm
2min, 94 DEG C of 30s, 60 DEG C of 30s, 68 DEG C of 4min, 30cycles, 68 DEG C of 10min), underlined sequences are restriction enzyme site.
PCR product is carried out together with plasmid mpNTKV-LoxP MAR using Sal I and Sac II after purification through PCR clean-up kit
Double digestion cuts out purpose band and recycles after agarose gel electrophoresis separates, and then carrier is connect with target fragment, then
Plasmid DNA is extracted in conversion to bacillus coli DH 5 alpha, picking single bacterium colony culture, is surveyed through digestion Preliminary Identification Hou Song commercial company
Sequence.Sequencing result display left side homology arm is inserted into successfully and is named as mpNTKV-LoxP MAL-MAR, and left side homology arm sequence is such as
Shown in SEQ ID NO.14 and Fig. 2.
Embodiment 3: the building of gene substitution targeting vector
From mpmCherry NMHC IIA (human wild type MYH9cDNA), mpEGFP NMHC IIB (human wild type
MYH10cDNA) or in mpEGFP NMHC IIA (mankind R702C point mutation MYH9cDNA) plasmid utilize Pac I and Pme I will
N-terminal merges the NMHC IIA of mCherry, and N-terminal merges NMHC IIB or R702C the NMHC IIA cDNA of GFP, and
SV40polyA expressed intact box cut out (these plasmids be this laboratory have been built up, mCherry or GFP5 ' end contain Pac I
Restriction enzyme site, SV40polyA 3 ' contain Pme I restriction enzyme site in end), while using Pac I and Swa I to mpNTKV-LoxP
MAL-MAR plasmid carries out double digestion.Digestion products cut out purpose band and recycle, then after agarose gel electrophoresis separates
Carrier is connect with target fragment, is then converted to bacillus coli DH 5 alpha, picking single bacterium colony culture, Plasmid DNA is extracted, through digestion
The sequencing of Preliminary Identification Hou Song commercial company.Sequencing result display substitution expression casette is successively inserted into left side homology arm and resistance
Between gene Neomycin, so far shows that complete MYH9 gene substitution targeting vector building finishes, be referred to as targeting vector
1,2,3, generated corresponding mutation allele is respectively designated as AmCh, Ab*, AgfpR702C, wild type is then A+, such as Fig. 2 institute
Show.
Each gene substitution targeting vector (100 μ g) is linearized using Not I, then uses UltraPurePhenol:
Chloroform:Isoamyl Alcohol (25:24:1, v/v) (Invitrogen) extracts DNA linearized enzyme digestion system twice,
Supernatant is transferred to another new 1.5ml EP after high speed centrifugation 10min to manage, 100% ethyl alcohol of 2.5 times of supernatant volumes is added, fills
Divide high speed centrifugation 10min after mixing to obtain white DNA pellet, is then precipitated once, in super-clean bench with 70% ethanol washing DNA
It removes supernatant and air-dries DNA and precipitate about 10min, after ethanol solution air-dries completely, it is small that appropriate TE solution dissolving DNA about 2 is added
When more than, measurement linearisation DNA concentration and to adjust DNA concentration be about 1 μ g/ μ l, while being examined using 0.8% agarose gel electrophoresis
DNA mass is surveyed, 4 DEG C of DNA solution save backup.
Embodiment 4: gene substitution targeting vector electricity is gone in ES cells
Anti- Neomycin (Neomycin-resistant) mouse embryonic fibroblasts (MEF) of gamma-radiation processing are used
Make trophocyte and V6.5 mice embryonic stem cell system (C57BL/6 and 129S6vEv heterozygosis genetic background) from US National
Cardiopulmonary Blood Research Institute transgenosis center.10cm Tissue Culture Dish with 3ml 0.1%Gelatin in 37 DEG C be coated with 2 hours it is spare,
Then 2X10 is added6Neomycin-resistant MEFs overnight incubation, is then inoculated with appropriate ES cell, in ES cell culture
Liquid is changed daily, it is long to be passed on to 70% abundance with 1:3 ratio.In 37 DEG C with 0.25% trypsin digestion cell 4min, blow and beat repeatedly to
Individual cells, and count, 1000rpm is collected by centrifugation, and PBS washed once, by 0.5X107A cell/ml concentration is resuspended in electricity and turns
In buffer (Millipore).It takes 0.6ml cell to be added in the 1.5ml EP pipe of the targeting vector DNA containing 50 μ g linearisation to mix,
And be transferred in the mating electric revolving cup of Eppendorf electroporation, it is stored at room temperature 5min.Turned with Eppendorf multiporator electricity
500 μ F electricity of instrument 320V turns, and takes out electric revolving cup and is placed at room temperature for 5min.It is transferred in the 50ml centrifuge tube of the culture solution containing 30ml and mixes,
It is evenly distributed again to being ready in 3 10cm culture dishes containing trophocyte in advance, 37 DEG C of 5%CO2It is cultivated in incubator.
It after 24 hours, changes into containing 800 μ g/ml G418 and 200 μM of ganciclovir culture solutions, hereafter the training of replacement drug containing daily
Nutrient solution.With special suction pipe picking embryonic stem cell monoclonal and prior standard is transferred under Nikon SMZ1500 microscope after a week
In 48 well culture plates containing trophocyte and culture solution got ready, in 37 DEG C of 5%CO2It is cultivated in incubator.After 24 hours,
The monoclonal cell of picking is digested with pancreatin and is blown and beaten 6 times, transfers to new preprepared containing trophocyte and training
In 48 well culture plates of nutrient solution, in 37 DEG C of 5%CO2It is cultivated 3-4 days in incubator, changes liquid daily.It is long extremely to monoclonal cell
1/3 cell suspension after cell is digested through pancreatin, blown and beaten, culture medium neutralizes, is transferred to preprepared containing taste by 80% abundance
In 24 well culture plates for supporting confluent monolayer cells and culture solution, and in 37 DEG C of 5%CO2Culture is used to Cell abundance up to 90% in incubator
In preparing genomic DNA；Isometric 2X embryonic stem cell frozen stock solution is added into the cell suspension for remaining in 48 well culture plates
(Millipore), culture plate surrounding is sealed with Parafilm and wrap up two layers or more blotting paper again, be stored in -80 DEG C of refrigerators for after
It is continuous to utilize.
The screening and identification of embodiment 5:MYH9 gene substitution embryonic stem cell monoclonal
The preparation of mouse embryo stem cell genomic DNA: the ES monoclonal through drug screening cultivated in 24 orifice plates is removed
The culture solution of cell, PBS washed once, and Nuclei lysis solution (Promega) of the 600 μ l containing RNaseA be added, instead
It blows and beats and is transferred in a clean EP pipe again, 200 μ l Protein precipitation solution are added, effectively
After Vortex half a minute, it is placed in and places 5min on ice, then high speed centrifugation 10min, transfer supernatant to another new EP is managed, be added etc.
The Isopropanol of volume and mixing after room temperature high speed centrifugation 10min, remove supernatant, heavy with 70% ethanol washing DNA of 1ml
It forms sediment primary, then after high speed centrifugation 5min, sucks supernatant with vacuum pump, be air-dried at room temperature 10-15min, be eventually adding 100 μ l DNA
Rehydration solution is stayed overnight and is saved backup in 4 DEG C of dissolutions.
The preparation of Southern Blot probe: forward primer LP-F5 '-is utilized by template of bMQ-369E5BAC DNA
GGATTGAACCTGAGGCTTTG (SEQ ID NO.15) and 5 '-GAGGAGGAGTGCTTGCTGTG (SEQ of reverse primer LP-R
ID NO.16) and Platinum Taq DNA Polymerase High Fidelity make probe on the left of PCR amplification about 1.2kb
(94℃ 2min,94℃ 30s,55℃ 30s,68℃ 80s,30cycles,68℃ 10min).PCR product is solidifying through agarose
After gel electrophoresis separation, cuts out purpose band and recycle, then connect target fragment with T-easy carrier (Promega), then
Plasmid DNA is extracted in conversion to bacillus coli DH 5 alpha, picking single bacterium colony culture, is surveyed through digestion Preliminary Identification Hou Song commercial company
Sequence.Sequencing result display left side probe, which constructs, is successfully named as LP.It is similar with the above method, using bMQ-369E5BAC DNA as mould
Plate utilizes 5 '-TTTACCCACTGACTCATACCTC of forward primer RP-F (SEQ ID NO.17) and reverse primer RP-R 5 '-
TGTGTTCTCCCTCCAATTACTC (SEQ ID NO.18) and Platinum Taq DNA Polymerase High
Fidelity make probe on the right side of PCR amplification about 1.6kb (94 DEG C of 2min, 94 DEG C of 30s, 55 DEG C of 30s, 68 DEG C of 100s,
30cycles,68℃ 10min).PCR product cuts out purpose band and recycles after agarose gel electrophoresis separates, and then will
Target fragment is connect with T-easy carrier, is then converted to bacillus coli DH 5 alpha, picking single bacterium colony culture, is extracted Plasmid DNA,
It is sequenced through digestion Preliminary Identification Hou Song commercial company.Sequencing result display right side probe, which constructs, is successfully named as RP.
Southern blot detection: utilize Dra I restriction enzyme (being purchased from Fermantas) by 10-15 μ g genome
DNA is stayed overnight in 37 DEG C of digestions, and 0.8% Ago-Gel 25V low-voltage electrophoresis separates overnight.DNA gel is respectively successively through 0.2N
After HCL, denaturation and neutralizer (Sigma) impregnate 20min, Nytran Supercharge Turboblotter is utilized
(Schleicher&Schuell Bioscience) and 20XSSC buffer (Sigma) carry out DNA marking transferring film, and transferring film finishes
Film is taken out afterwards and impregnates 10min in 2XSSC solution, is crosslinked followed by UV light.At the same time, EcoR I is utilized
(Fermantas) DNA probe is cut out from T-easy carrier and DNA glue recovery purifying is done to probe fragment, utilize Ready-to-
Go DNA Labeling Beads (Amersham) and32P dCTP is to probe in 37 DEG C of progress isotope labelling 10min, label
Probe is purified using ProbeQuant G50Micro Columns (Amersham) afterwards.It will turn the film for having genomic DNA
It is put into hybrid pipe, 30ml Hybrisol I (Millipore) prehybridization 30min in 42 DEG C of hybrid heaters is added, is then added
The probe of isotope labelling and 100 μ l Solmon Sperm DNA (Sigma), then the hybridized overnight in hybrid heater, takes out miscellaneous
After handing over film and being washed three times with 1XSSC+0.1%SDS, is stayed overnight in -80 DEG C of exposure pieces, finally develop a film and obtain result.According to above-mentioned side
Method carries out Southern Blot, A using left side probe+, AmCh,Ab*And AgfpR702CThe expection stripe size of each allele is distinguished
For 9.7kb, 12.1kb, 8.5kb, 12.1kb.Southern Blot, A are carried out using right side probe+, AmCh,Ab*And AgfpR702CRespectively
The expection stripe size of allele is respectively 9.7kb, 6.0kb, 6.0kb, 6.0kb, as shown in Figure 3.Southern Blot inspection
Survey shows each gene substitution target practice homologous recombination efficiency more than 90%, and only MYH9 allele mutates, tool
The results are shown in Table 1 for body.
Claims (4)
1. a kind of method for constructing gene substitution mice study wild type and the non-flesh myoglobulin heavy chain II function of mutant,
It is characterized in that, described method includes following steps:
(1) mpNTKV-LoxP carrier is constructed, the mpNTKV-LoxP carrier sequence is as shown in SEQ ID NO.4；
(2) targeting vector of targeting MYH9 gene is constructed on the basis of mpNTKV-LoxP carrier: by mouse MYH9 gene extron
2 upstream 4.0kb and downstream 1.7kkb DNA sequence dna are inserted into mpNTKV-LoxP carrier the target practice for obtaining targeting MYH9 gene respectively
Carrier is named as mpNTKV-LoxP MAL-MAR targeting vector, and the mpNTKV-LoxP MAL-MAR targeting vector sequence is such as
Shown in SEQ ID NO.25；
(3) cDNA of the non-flesh myoglobulin heavy chain II of the coding fusion fluorescin of the quasi- endogenous MYH9 gene of substitution is distinguished
It is described different to obtain three different MYH9 gene substitution targeting vectors in insertion targeting MYH9 gene targeting carrier
MYH9 gene substitution targeting vector is respectively designated as targeting vector 1, targeting vector 2, targeting vector 3；The sequence of the targeting vector 1
It arranges as shown in SEQ ID NO.26, the sequence of the targeting vector 2 is as shown in SEQ ID NO.27, the sequence of the targeting vector 3
Column are as shown in SEQ ID NO.28；
(4) the MYH9 gene substitution targeting vector of building is transduceed in ES cells by electricity, is obtained carefully through drug screening
Born of the same parents' monoclonal through Southern Blot or PCR identification of cell monoclonal, and then obtains MYH9 gene substitution ES cell line, described
MYH9 gene substitution ES cell line is for generating MYH9 gene substitution mouse.
2. the method as described in claim 1, which is characterized in that drug screening described in step (4) is through 418 He of G
Ganciclovir drug screening.
3. a kind of new support, which is characterized in that the carrier is to construct to obtain based on pNTKV1905 carrier, the new support
Sequence is named as mpNTKV-LoxP carrier as shown in SEQ ID NO.4.
4. a kind of targeting vector, which is characterized in that the targeting vector sequence is named as shown in SEQ ID NO.25
MpNTKV-LoxP MAL-MAR targeting vector.
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| CN103993027A (en) * | 2014-04-17 | 2014-08-20 | 中国农业大学 | Transgenic pig screening marker gene knockout method |
| WO2016049163A2 (en) * | 2014-09-24 | 2016-03-31 | The Broad Institute Inc. | Use and production of chd8+/- transgenic animals with behavioral phenotypes characteristic of autism spectrum disorder |
| CN106434750A (en) * | 2016-09-26 | 2017-02-22 | 湖南农业大学 | Target targeting vector, method for constructing mouse embryonic stem cell strain through targeting integration of exogenous genes to MYH9 Intron2 locus and application |
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| CN103993027A (en) * | 2014-04-17 | 2014-08-20 | 中国农业大学 | Transgenic pig screening marker gene knockout method |
| WO2016049163A2 (en) * | 2014-09-24 | 2016-03-31 | The Broad Institute Inc. | Use and production of chd8+/- transgenic animals with behavioral phenotypes characteristic of autism spectrum disorder |
| CN106434750A (en) * | 2016-09-26 | 2017-02-22 | 湖南农业大学 | Target targeting vector, method for constructing mouse embryonic stem cell strain through targeting integration of exogenous genes to MYH9 Intron2 locus and application |
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