CN105400810B - A method for establishing a hypophosphatemic rickets model by knockout technology - Google Patents
A method for establishing a hypophosphatemic rickets model by knockout technology Download PDFInfo
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Abstract
A method of phosphopenic rickets model is established using knockout technology, belongs to field of biotechnology.The purpose of the present invention is the method for establishing phosphopenic rickets model using knockout technology of mankind's phosphopenic rickets model is successfully obtained using CRISPR-CAS9 gene Knockout.Step of the invention is: the building of sgRNA expression vector, the synthesis of CAS9mRNA, the acquisition and microinjection of fertilized eggs, the culture and development of fertilized eggs.The present invention successfully obtains phosphopenic rickets model, the model obtains the pathologic process for capableing of effective simulation human diseases, it can more effectively predict the effect in clinical application such as novel vaccine, new drug and new diagnostic reagent, the risk for substantially reducing new drug development simultaneously, provides basic model for clinical research.
Description
Technical field
The invention belongs to field of biotechnology.
Background technique
Phosphopenic rickets (Hypophosphatemic rickets, HR) are one group because kidney phosphorus loss leads to serium inorganic phosphorus
It reduces, causes the disease of pathological mineralization.It is divided into four kinds of hypotypes, and clinically the chain dominant phosphopenic rickets (XLH) of X are most
Common, disease incidence is about 1/20000, and it is short and small to can lead to patient size, ostalgia, development abnormalities of teeth, lower limb malformation, and
There is rickets etc. in children.Caused by the cause of disease of XLH is PHEX gene Inactivating mutations.
The illness model of human diseases is the important foundation of pathogenic mechanism research and new drug development.Therefore human diseases is established
Model, the pathologic process of effective simulation human diseases can more effectively predict that novel vaccine, new drug and new diagnostic reagent etc. are facing
Effect in bed application, while substantially reducing the risk of new drug development.
Summary of the invention
The purpose of the present invention is successfully obtain mankind's phosphopenic rickets model using CRISPR-CAS9 gene Knockout
The method that phosphopenic rickets model is established using knockout technology.
Step of the invention is:
1. the building of sgRNA expression vector: designing 2 sgRNA sequences at the 1st exon of PHEX gene and act on target
Point synthesizes two couples of oligonucleotide chain preparation sgRNA, wherein two pairs of oligonucleotides are as follows:
SgRNA1:TAGGCAATTCGAGTGCCTCTGG and AAACCCAGAGGCACTCGAATTG；
SgRNA2:TAGGCTTGGCACGATCCTCTTTC and AAACGAAAGAGGATCGTGCCAAG；
The oligonucleotide of synthesis is annealed, and annealing conditions are: being down to room temperature naturally after 95 DEG C of 5min, is connected into through Bbs I
The recovered PUC57-sgRNA expression vector of digestion completes sgRNA vector construction；
Wherein: digestion system: 20 μ l of plasmid PUC57
10×buffer 20μl
BbsⅠ 1μl
ddH2O 159μl
37 DEG C of 3h of digestion are recycled after electrophoresis runs glue using common DNA agarose gel QIAquick Gel Extraction Kit；
2. the synthesis of CAS9mRNA: CAS9 expression plasmid is through linearization for enzyme restriction, after purification through phenol chloroform, is dissolved in seedless
In the water of sour enzyme；
Digestion system: I 4 μ l of Not
CAS9 50μl
BSA 30μl
Triton 30μl
10×H 30μl
ddH2O 156μl
37 DEG C of 3h of digestion are recycled after electrophoresis runs glue using common DNA agarose gel QIAquick Gel Extraction Kit；
3. the acquisition and microinjection of fertilized eggs: injection follicular stimulating hormone is injected human chorionic gonadotrophin later, is obtained
Fertilized eggs are taken, pre- mixed CAS9mRNA/sgRNA mixture is injected into cytoplasm by microinjection instrument, wherein
CAS9mRNA concentration is that 150ng/ μ l, sgRNA concentration is 30ng/ μ l；
4. the culture and development of fertilized eggs: the fertilized eggs of microinjection being transferred in culture solution, 37 DEG C of constant temperature trainings are placed in
It supports and is cultivated in case, when development is to mulberry body period, with inhaling ovum needle for single embryo transfer into centrifuge tube.
The widow that the oligonucleotide chain selection principle of sgRNA of the present invention: choosing score highest and base sequence beginning is GG
Polynucleotide chain.
Embryo PHEX gene knockout situation identification method of the present invention:
1. embryo cracks: embryo's lytic reagent is NP40, cracking condition are as follows: 56 DEG C, 1h；95 DEG C, 10min；
2. DNA sequencing identifies that embryonic gene type knocks out situation: extracting DNA, carry out PCR, electroresis appraisal, and carry out DNA survey
Sequence obtains genotype identification result；
A, the designed PCR primers as follows:
Upstream primer: GTCAACGCTTAACAGACTC
Downstream primer: CACCTCCAACCACTTAATAC；
B, PCR reaction system is as follows:
Template DNA 1ul
Upstream primer 1ul
Downstream primer 1ul
2×Taq plus 12.5ul
ddH2O 9.5ul；
C, PCR reaction condition:
95 DEG C of initial denaturation 7min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30 s, 72 DEG C of extension 40s；30 circulations；72 ℃
Extend 5min；
3. PCR product is sequenced, sequencing result occurs bimodal in the target practice location proximate that PHEX gene primer designs
Situation, selects bimodal sample PCR again, is attached PGM-T carrier after the recycling of product glue, and picking positive colony is again after conversion
It is secondary to be sequenced, base insertion or base deletion occurs in sequencing result near PHEX gene target site, causes to read and frame shift
Code mutation, then be gene knockout.
The present invention passes through coherent detection, successfully obtains phosphopenic rickets model, and the acquisition of the model being capable of effectively mould
The pathologic process of quasi- human diseases, can more effectively predict the effect in clinical application such as novel vaccine, new drug and new diagnostic reagent
Fruit, while the risk of new drug development is substantially reduced, basic model is provided for clinical research.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of expression vector PUC57-sgRNA of the present invention；
Fig. 2 is the electrophoretogram of PCR product identification embryo PHEX gene knockout situation of the present invention；
Wherein M:MarkerFor DNA molecular standard volume；1-14: 14 embryo's DNA PCR results after microinjection.15:
Positive control (normal fetus)；16: water control；
The PHEX gene of design identifies that primer is 448bp
It is available from DNA sequencing result and PCR product electrophoresis result: 1,2,3,4,5,6,7,8,9,10,11,12,13,
Different knockout situations occurs for 14,15 embryos；
Fig. 3 is that control group and knockout group are recorded and counted respectively after the newborn rabbit that obtains after microinjection is identified
Its situation that body is grown during the growth process；
The figure is normal group and knockout group rabbit growth curve, can be seen from the chart that knockout group goes out in growth and development process
Existing growth retardation phenomenon；
Fig. 4 is that control group and knockout group are recorded and counted respectively after the newborn rabbit that obtains after microinjection is identified
Its during the growth process weight the case where；
The figure is normal group and knockout group rabbit height curve, it can be seen that knockout group height be significantly lower than normal group, and
Growth retardation；
Fig. 5 is that young rabbit anteserum qualification result is obtained after microinjection: content of alkaline phosphatase comparison；The chart reveals:
Normal group compares with knockout group, and the content of knockout group alkaline phosphatase significantly increases；
Fig. 6 is that young rabbit anteserum qualification result is obtained after microinjection: phosphorus content comparison；The chart reveals: normal group and
Knockout group compares, and knockout group phosphorus content significantly lowers.
Specific embodiment
The present invention establishes PHEX gene knockout phosphopenic rickets model:
Steps are as follows:
1) building of CRISPR-CAS9 system sgRNA design and expression vector.
According to Dr. Feng Zhang(http: //crispr.mit.edu/) it designs at the 1st exon of PHEX gene
2 sgRNA sequence action target spots are designed, synthesize two pairs
Oligonucleotide chain (sgRNA1:TAGGCAATTCGAGTGCCTCTGG and AAACCCAGAGGCACTCGAATTG;
SgRNA2:TAGGCTTGGCACGATCCTCTTTC and AAACGAAAGAGGATCGTGCCAAG) it is used to prepare sgRNA.The sgRNA
Oligonucleotide chain selection principle: choose score highest and base sequence beginning be GG oligonucleotide chain.The widow of synthesis
Polynucleotide is annealed (being down to room temperature naturally after 95 DEG C of 5min), is connected into through the recovered PUC57-sgRNA table of I digestion of Bbs
Up to carrier, sgRNA vector construction is completed, is connected correctly, is cloned by sequence verification segment, extracts plasmid after expanding culture
For preparing that template is transcribed in vitro.
Digestion system: 20 μ l of plasmid PUC57
10×buffer 20μl
BbsⅠ 1μl
ddH2O 159μl
37 DEG C of 3h of digestion after electrophoresis runs glue, (are purchased from Tiangeng company, north using common DNA agarose gel QIAquick Gel Extraction Kit
Capital, China) it is recycled, concrete operations by specification carries out.
CAS9 expression plasmid (Addgene, laboratory purchase), through linearization for enzyme restriction, after purification through phenol chloroform, is dissolved in
Template is used as in the water of nuclease free, for being transcribed in vitro.The synthesis of CAS9mRNA is by kit RNeasy Mini Kit
(Qiagen, No.74104) acts on t7 rna polymerase in vitro to complete, and the external synthesis of sgRNA is by kit MiRNeasy
Mini Kit (Qiasgen, No.217004) is completed using t7 rna polymerase in vitro.
Digestion system: I 4 μ l of Not
CAS9 50μl
BSA 30μl
Triton 30μl
10×H 30μl
ddH2O 156μl
37 DEG C of 3h of digestion after electrophoresis runs glue, (are purchased from Tiangeng company, north using common DNA agarose gel QIAquick Gel Extraction Kit
Capital, China) it is recycled, concrete operations by specification carries out.
2) acquisition and microinjection of fertilized eggs.
It injects follicular stimulating hormone (FSH), injects human chorionic gonadotrophin (HCG) later and (be purchased from the second hormone of Ningbo
Factory), fertilized eggs are obtained, pre- mixed CAS9mRNA/sgRNA mixture is injected into cytoplasm by microinjection instrument
(final concentration of 30ng/ μ l of final concentration of 150ng/ the μ l, sgRNA of CAS9mRNA).
3) in vitro culture and development of fertilized eggs.
The fertilized eggs of microinjection are transferred in culture solution, is placed in 37 DEG C of constant incubators and cultivates, mulberry fruit is arrived in development
When embryo period, with inhaling ovum needle by single embryo transfer into centrifuge tube, with testing later.
(2) embryo PHEX gene knockout situation is identified
1) embryo cracks
Embryo's lytic reagent is NP40, cracking condition are as follows: 56 DEG C of 1h
95℃ 10min
2) DNA sequencing identification embryonic gene type knocks out situation.
Extract DNA, extracting method operated according to tissue gene group extracts kit specification (be purchased from Tiangeng company,
Beijing, China), PCR, electroresis appraisal are carried out, and carry out DNA sequencing, obtains genotype identification result.
The designed PCR primers as follows:
Upstream primer: GTCAACGCTTAACAGACTC
Downstream primer: CACCTCCAACCACTTAATAC
PCR reaction system is as follows:
Template DNA 1ul
Upstream primer 1ul
Downstream primer 1ul
2×Taq plus 12.5ul
ddH2O 9.5ul
PCR reaction condition:
95 DEG C of initial denaturation 7min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30 s, 72 DEG C of extension 40s；30 circulations；72 ℃
Extend 5min.
PCR product is sequenced, if sequencing result occur in the target practice location proximate that PHEX gene primer designs it is bimodal
Situation, then to practice shooting successfully.Bimodal sample PCR again is selected, PGM-T carrier is attached after the recycling of product glue, after conversion
Picking positive colony is sequenced again, and base insertion occurs near PHEX gene target site in sequencing result or base lacks
It loses, leads to frame shift mutation, be then judged as gene knockout.
Technical solution of the present invention is clearly and completely described below:
(1) it establishes rabbit PHEX and knocks out phosphopenic rickets model.
Steps are as follows :-
1) building of CRISPR-CAS9 system sgRNA design and expression vector.
2 sgRNA sequence action target spots are designed at the 1st exon of PHEX gene, synthesize two pairs
Oligonucleotide chain (sgRNA1:TAGGCAATTCGAGTGCCTCTGG and AAACCCAGAGGCACTCGAATTG;
SgRNA2:TAGGCTTGGCACGATCCTCTTTC and AAACGAAAGAGGATCGTGCCAAG) it is used to prepare sgRNA.The sgRNA
Oligonucleotide chain selection principle: choose score highest and base sequence beginning be G oligonucleotide chain.The oligomerization of synthesis
Nucleotide is annealed (being down to room temperature naturally after 95 DEG C of 5min), is connected into through the recovered PUC57-sgRNA expression of I digestion of Bbs
Carrier completes sgRNA vector construction, is connected correctly, is cloned by sequence verification segment, extracts plasmid after expansion culture and uses
Template is transcribed in vitro in preparation.
Digestion system: 20 μ l of plasmid PUC57
10×buffer 20μl
BbsⅠ 1μl
ddH2O 159μl
37 DEG C of 3h of digestion after electrophoresis runs glue, (are purchased from Tiangeng company, north using common DNA agarose gel QIAquick Gel Extraction Kit
Capital, China) it is recycled, concrete operations by specification carries out.
CAS9 expression plasmid (Addgene, laboratory purchase), through linearization for enzyme restriction, after purification through phenol chloroform, is dissolved in
Template is used as in the water of nuclease free, for being transcribed in vitro.The synthesis of CAS9mRNA is by kit RNeasy Mini Kit
(Qiagen, No.74104) acts on t7 rna polymerase in vitro to complete, and the external synthesis of sgRNA is by kit MiRNeasy
Mini Kit (Qiasgen, No.217004) is completed using t7 rna polymerase in vitro.
Digestion system: I 4 μ l of Not
CAS9 50μl
BSA 30μl
Triton 30μl
10×H 30μl
ddH2O 156μl
37 DEG C of 3h of digestion after electrophoresis runs glue, (are purchased from Tiangeng company, north using common DNA agarose gel QIAquick Gel Extraction Kit
Capital, China) it is recycled, concrete operations by specification carries out.
2) acquisition and microinjection of fertilized eggs.
It injects follicular stimulating hormone (FSH), injects human chorionic gonadotrophin (HCG) later and (be purchased from the second hormone of Ningbo
Factory), fertilized eggs are obtained, pre- mixed CAS9mRNA/sgRNA mixture is injected into cytoplasm by microinjection instrument
(final concentration of 30ng/ μ l of final concentration of 150ng/ the μ l, sgRNA of CAS9mRNA).
3) zygote transplation and animal after injecting are cultivated.
After microinjection, by zygote transplation, embryonic development is carried out, standardization raising is carried out.
(2) rabbit phosphopenic rickets model phenotypic evaluation and genotyping.
1) rabbit phenotypic results count.
1 week after birth, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, progress rabbit appearance photo acquisition in 12 weeks.
2) genotype of DNA sequencing identification rabbit phosphopenic rickets model.
Extract tissue DNA, extracting method operated according to tissue gene group extracts kit specification (Tiangeng, Beijing,
China), PCR, electroresis appraisal are carried out, and carry out DNA sequencing, obtains genotype identification result.
The designed PCR primers as follows:
Upstream primer: GTCAACGCTTAACAGACTC
Downstream primer: CACCTCCAACCACTTAATAC
PCR reaction system is as follows:
Template DNA 1ul
Upstream primer 1ul
Downstream primer 1ul
2×Taq plus 12.5ul
ddH2O 9.5ul
PCR reaction condition:
95 DEG C of initial denaturation 7min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30 s, 72 DEG C of extension 40s；30 circulations；72 ℃
Extend 5min.
PCR product is sequenced, if sequencing result occur in the target practice location proximate that PHEX gene primer designs it is bimodal
Situation then may be to practice shooting successfully.Bimodal sample PCR again is selected, carrier T is attached after the recycling of product glue, after conversion
Picking positive colony is sequenced again, if occurring near PHEX gene target site in sequencing result, base is inserted into or base lacks
It loses, leads to frame shift mutation, then can determine whether as gene knockout.
3) coherent detection is carried out in mRNA level in-site using fluorescence quantifying PCR method.
Design Q-PCR primer:
GAPDH: upstream primer ATCCATTCATTGACCTCCACTAC
Downstream primer GTACTGGGCACCAGCATCAC
FGF23: upstream primer CACACCTCATCAGACCATCTAC
Downstream primer TGTTGCCTCTGAAATCCATACA
SOST: upstream primer CAGCCGTCCACATGGTAGAG
Downstream primer GCTGTACTCCGACACGTCTTT
PTH: upstream primer GTCTGCTCAAGACATGGCTAA
Downstream primer GAGGAACTCTGAGGCCAATAAA
The extraction of rabbit kidney RNA: will freeze in the renal tissue of -80 DEG C of rabbits, ground using liquid nitrogen, use
TRNzol-A+Total RNA extraction reagent box extracts, operation be carried out according to kit specification (be purchased from Tiangeng company, Beijing,
China).
Reverse transcription: the RNA that upper step is extracted carries out reverse transcription, using FastQuant cDNA the first chain synthetic agent
Box (Tiangeng, Beijing, China), operation carries out to specifications.
Using BioEasySYBR Green I Real-Time PCR Kit carry out quantitative analysis, the present invention choose FGF23,
The genes such as SOST, PTH carry out quantitative analysis, reference gene GAPDH.
4) analysis of PHEX genes protein level is carried out using immunoblotting (Western Blot) method.
Specific step is as follows by Western Blot:
Preparation of samples takes 10ug protein sample first, and sample-loading buffer is added, and boiling water is denaturalized 5 minutes, is put into immediately
On ice, 12000rpm, 4 DEG C are centrifuged 5 minutes.
Electrophoresis: protein sample being loaded in the sample well of the poly amic acid gel prepared, carries out electrophoresis.
Electricity turns: shifting " sandwich " according to the sequentially built of foam-rubber cushion, filter paper, film, gel, filter paper, foam-rubber cushion, is put into
In electric turn trough, 100V voltage electricity turns 3 hours.
It washes film: taking out film, be put in TBST and wash 15 minutes, continuously wash 3 times.
Closing: film is put in the TBST solution of 5% skimmed milk and is closed 2 hours.
Primary antibody is incubated for: carrying out primary antibody dilution, 4 DEG C of overnight incubations with the TBST containing 5% skimmed milk.
It washes film: film being washed 15 minutes with TBST, continuous 3 times.
Secondary antibody: secondary antibody dilution is carried out with the TBST containing 5% skimmed milk, is incubated for 2 hours.
It washes film: film being washed 15 minutes with TBST, continuous 3 times.
Development: it needs to develop with developer solution using visualizer.
(3) rabbit phosphopenic rickets model phenotypic evaluation and genotyping.
1) rabbit body length and weight results acquisition.
1 week after birth, measures normal rabbits and knock out the body length and body of rabbit within 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks
Weight.
2) rabbit related serological result measures.
Respectively at 7 weeks, 14 weeks progress determination of serology, serum is separated, using phosphorus, alkali in ELISA kit measurement serum
The biochemical indicators such as acid phosphatase, calcium.
Separation serological method: taking new blood, is put in the centrifuge tube of 1.5ml sterilizing, and 37 DEG C stand 30 minutes,
3000rpm is centrifuged 10 minutes, and faint yellow supernatant is drawn onto centrifuge tube, -80 DEG C of preservations are put in, and is used for experimental study.
3) vitals such as rabbit bone, heart, liver, spleen, kidney, lungs are observed or whether tissue occurs lesion.
It knocks out rabbit during the growth process, dead individual occurs, each organ disease situation is dissected and observed, fixing organization is done
Tissue pathological slice.
4) credit of rabbit skeletal image is analysed.
It at the 7th week, takes and rejects clean appendicular skeleton, carry out X-ray, obtain appendicular skeleton iconography photo, carry out phase
Answer interpretation of result.
1, the sequence of PHEX gene First Exon:
ATGGAAGCAGAAACAGGGAGCAATGCGGGAACTGGAAAGACGGCCACCAGAGGCAC
TCGAATTGCCCTGGTCGTATTCATCGGCGGCACTCTCGTGCTTGGCACGATCCTCTTTCT
GG
2, normal fetus PCR product carries out the result (length 448bp) of DNA sequencing:
GTCAACGCTTAACAGACTCTTGAGAGCAGCCACCAGACCACGAAAAGTGACTAAGATTCTTCTCGTGTG
CTCTCTACGGC
CCTTCTGATGGAAGCAGAAACAGGGAGCAATGCGGGAACTGGAAAGACGGCCACCAGAGGCACTCGAAT
TGCCCTGGTCG
TATTCATCGGCGGCACTCTCGTGCTTGGCACGATCCTCTTTCTGGGTAAGTGGAGCCCTGGAGGCCGGG
GGCACGGGGCA
TGTGCTGAGAAATCCCTTGTGCTTTATTCTAGCCTTGTCATAATGCAAGTGTGCGACTGTTTGCTTGTG
TTAGAAGGTGA
TGCAGTCTTGGTTTCTATCAAATGGGCAAATGAAAAAGGCTTTGCTTTCAGAGGCTACATGCATAAATA
CAGTCAAGAAC
TGAATATGTACTGCAGAGGGCAAAGTGGGTATTAAGTGGTTGGAGGT
3, PUC57-sgRNA1 sequence (italic underscore is labeled as mono- oligonucleotide chain of sgRNA1):
CAGTGATTGGAGATCGGTACTTCGCGAATGCGTCGAGATATTGGGTCTTTAAAAGCACCGACTCGGTGC
CACTTTTTCAA
GTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTA AAACCCAGAGGCACTCGAATTG C
CTATAGTGAGT
CGTATTAATTGGGTATCGGATGCCGGGACCGACGAGTGCAGAGGCGTGCAAGCGAGCTTGGCGTAATCA
TGGTCATAGCT
GTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAA
AGCCTGGGGTG
CCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGT
CGTGCCAGCTG
CATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTC
ACTGACTCGCT
GCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGA
ATCAGGGGATA
ACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG
CGTTTTTCCAT
AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGA
CTATAAAGATA
CCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCT
GTCCGCCTTTC
TCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTC
GCTCCAAGCTG
GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGGTAACTATCGTCTTGAGTC
CAACCCGGTAA
GACACGACTTATCGCCACTGGCAGCAGCCACTGGTACAGGATTAGCAGAGCGAGTATGTAAGCGTTGCT
ACAGAGTTCTT
GAAGTGGTGCCTAACTACGGGCTTACCACTAAGGA
4, PUC57-sgRNA2 sequence (italic underscore is labeled as mono- oligonucleotide chain of sgRNA2):
CGGCAGTGATTGGAGATCGGTACTTCGCGAATGCGTCGAGATATTGGGTCTTTAAAAGCACCGACTCGG
TGCCACTTTTT
CAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTA AAACGAAAGAGGATCGTGCC AAG CCTATAGT
GAGTCGTATTAATTGGGTATCGGATGCCGGGACCGACGAGTGCAGAGGCGTGCAAGCGAGCTTGGCGTA
ATCATGGTCAT
AGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGT
GTAAAGCCTGG
GGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAAC
CTGTCGTGCCA
GCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTC
GCTCACTGACT
CGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCA
CAGAATCAGGG
GATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTG
CTGGCGTTTTT
CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGAC
AGGACTATAAA
GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT
ACCTGTCCGCC
TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTC
GTTCGCTCCAA
GCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGA
GTCCAACCCGG
TAAGACACGACTTATCGCACTGGCAGCAGCACTGTAACAGATTAGCAGAGCGAGGTATGTAGGCGGTGC
TACAGAGT
Claims (3)
1. a kind of method for establishing phosphopenic rickets model using knockout technology, it is characterised in that:
1. the building of sgRNA expression vector: designing 2 sgRNA sequence action target spots at the 1st exon of PHEX gene, close
SgRNA is prepared at two pairs of oligonucleotide chains, wherein two pairs of oligonucleotides are as follows:
SgRNA1:TAGGCAATTCGAGTGCCTCTGG and AAACCCAGAGGCACTCGAATTG；
SgRNA2:TAGGCTTGGCACGATCCTCTTTC and AAACGAAAGAGGATCGTGCCAAG；
The oligonucleotide difference of synthesis is annealed, and annealing conditions are: being down to room temperature naturally after 95 DEG C of 5min, uses Bbs I respectively
Digestion products are then recycled PUC57 vector linearization by restriction endonuclease, and by sgRNA1 and sgRNA2 points
It is not connected on PUC57 carrier, and then completes the building of PUC57-sgRNA1 and PUC57-sgRNA2 carrier；
Wherein: digestion system: 20 μ l of plasmid PUC57
10×buffer 20μl
BbsⅠ 1μl
ddH2O 159μl
37 DEG C of 3h of digestion are recycled after electrophoresis runs glue using common DNA agarose gel QIAquick Gel Extraction Kit；
2. the synthesis of CAS9mRNA: CAS9 expression plasmid is through linearization for enzyme restriction, after purification through phenol chloroform, is dissolved in nuclease free
Water in；
Digestion system: I 4 μ l of Not
CAS9 50μl
BSA 30μl
Triton 30μl
10×H 30μl
ddH2O 156μl
37 DEG C of 3h of digestion are recycled after electrophoresis runs glue using common DNA agarose gel QIAquick Gel Extraction Kit；
3. the acquisition and microinjection of fertilized eggs: injection follicular stimulating hormone injects human chorionic gonadotrophin later, obtain by
Pre- mixed CAS9mRNA/sgRNA mixture is injected into cytoplasm, wherein CAS9mRNA by smart ovum by microinjection instrument
Concentration is that 150ng/ μ l, sgRNA concentration is 30ng/ μ l；
4. the culture and development of fertilized eggs: the fertilized eggs of microinjection being transferred in culture solution, 37 DEG C of constant incubators are placed in
Middle culture, when development is to mulberry body period, with inhaling ovum needle for single embryo transfer into centrifuge tube.
2. the method according to claim 1 for establishing phosphopenic rickets model using knockout technology, it is characterised in that:
The oligonucleotide chain that the oligonucleotide chain selection principle of sgRNA: choosing score highest and base sequence beginning is GG.
3. the method described in claim 1 for establishing phosphopenic rickets model using knockout technology, it is characterised in that: further include
Embryo's PHEX gene knockout situation identification method:
1. embryo cracks: embryo's lytic reagent is NP40, cracking condition are as follows: 56 DEG C, 1h；95 DEG C, 10min；
2. DNA sequencing identifies that embryonic gene type knocks out situation: extracting DNA, carry out PCR, electroresis appraisal, and carry out DNA sequencing, obtain
To genotype identification result；
A, the designed PCR primers as follows:
Upstream primer: GTCAACGCTTAACAGACTC
Downstream primer: CACCTCCAACCACTTAATAC；
B, PCR reaction system is as follows:
Template DNA 1ul
Upstream primer 1ul
Downstream primer 1ul
2×Taq plus 12.5ul
ddH2O 9.5ul；
C, PCR reaction condition:
95 DEG C of initial denaturation 7min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30 s, 72 DEG C of extension 40s；30 circulations；72 DEG C of extensions
5min；
3. PCR product is sequenced, there is bimodal situation in the target practice location proximate that PHEX gene primer designs in sequencing result,
Select bimodal sample PCR again, be attached PGM-T carrier after the recycling of product glue, after conversion picking positive colony again into
Row is sequenced, and base insertion or base deletion occurs in sequencing result near PHEX gene target site, causes reading frame frameshit prominent
Become, is then gene knockout.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510557129.7A CN105400810B (en) | 2015-09-06 | 2015-09-06 | A method for establishing a hypophosphatemic rickets model by knockout technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510557129.7A CN105400810B (en) | 2015-09-06 | 2015-09-06 | A method for establishing a hypophosphatemic rickets model by knockout technology |
Publications (2)
| Publication Number | Publication Date |
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