CN105400810A - Method for establishing phosphopenic rickets model by using knockout technology - Google Patents
Method for establishing phosphopenic rickets model by using knockout technology Download PDFInfo
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Abstract
The invention relates to a method for establishing a phosphopenic rickets model by using a knockout technology, and belongs to the technical field of biology. A purpose of the present invention to provide a method for establishing a phosphopenic rickets model by using a knockout technology, wherein a human phosphopenic rickets model is successfully obtained by using a CRISPR-CAS9 gene knockout technology. The method comprises sgRNA expression vector constructing, CAS9mRNA synthesis, oosperm acquisition and microinjection, and oosperm culture and development. According to the present invention, the phosphopenic rickets model is successfully obtained; and wit the obtaining of the phosphopenic rickets model, the pathological process of human disease can be effectively simulated, the effects of new vaccines, new drugs, new diagnostic reagents and the like in clinical applications can be effectively predicted, the new drug development risk can be substantially reduced, and the base model can be provided for clinical research.
Description
Technical field
The invention belongs to biological technical field.
Background technology
Phosphopenic rickets (Hypophosphatemicrickets, HR) be one group because of kidney phosphorus lose cause serium inorganic phosphorus to reduce, cause the disease of pathological mineralization.It is divided into four kinds of hypotypes, and the chain dominant phosphopenic rickets of X (XLH) is the most common clinically, and its sickness rate is about 1/20000, patient can be caused of short and small stature, ostalgia, development abnormalities of teeth, lower limb malformation, and rickets etc. appears in children.The cause of disease of XLH is that PHEX gene inactivation sudden change causes.
The illness model of human diseases is the important foundation of pathogenic mechanism research and new drug development.Therefore set up human disease model, effectively the pathologic process of simulating human disease, more effectively can predict the effect in clinical application such as novel vaccine, new drug and new diagnostic reagent, greatly reduce the risk of new drug development simultaneously.
Summary of the invention
The employing that the object of the invention is to adopt CRISPR-CAS9 gene Knockout successfully to obtain mankind's phosphopenic rickets model knocks out the method that technology sets up phosphopenic rickets model.
Step of the present invention is:
1. the structure of sgRNA expression vector: the 1st exon place designs 2 sgRNA sequence action target spots at PHEX gene, synthesize two pairs of oligonucleotide chains and prepare sgRNA, wherein two pairs of oligonucleotides are:
SgRNA1:TAGGCAATTCGAGTGCCTCTGG and AAACCCAGAGGCACTCGAATTG;
SgRNA2:TAGGCTTGGCACGATCCTCTTTC and AAACGAAAGAGGATCGTGCCAAG;
The oligonucleotide of synthesis is through annealing, and annealing conditions is: be naturally down to room temperature after 95 DEG C of 5min, is connected into and cuts through reclaiming PUC57-sgRNA expression vector through Bbs I enzyme, complete sgRNA vector construction;
Wherein: enzyme cuts system: plasmid PUC5720 μ l
10×buffer20μl
BbsⅠ1μl
ddH
2O159μl
Enzyme cuts 37 DEG C of 3h, after electrophoresis runs glue, uses common DNA agarose gel to reclaim test kit and reclaims;
2. the synthesis of CAS9mRNA: CAS9 expression plasmid, through linearization for enzyme restriction, after phenol chloroform purifying, is dissolved in the water of nuclease free;
Enzyme cuts system: Not I 4 μ l
CAS950μl
BSA30μl
Triton30μl
10×H30μl
ddH
2O156μl
Enzyme cuts 37 DEG C of 3h, after electrophoresis runs glue, uses common DNA agarose gel to reclaim test kit and reclaims;
3. the acquisition of zygote and microinjection: injection follicular stimulating hormone, inject human chorionic gonadotrophin afterwards, obtain zygote, by microinjection instrument, pre-mixed CAS9mRNA/sgRNA mixture is expelled in tenuigenin, wherein CAS9mRNA concentration is 150ng/ μ l, and sgRNA concentration is 30ng/ μ l;
4. the cultivation of zygote and growth: transfer in nutrient solution by the zygote of microinjection, be placed in 37 DEG C of constant incubators and cultivate, when growing to morula period, transfers to single embryo in centrifuge tube with suction ovum pin.
The oligonucleotide chain selection principle of sgRNA of the present invention: choose the oligonucleotide chain that the beginning of mark the highest and base sequence is GG.
Embryo PHEX gene knockout situation authentication method of the present invention:
1. embryo's cracking: embryo's lytic reagent is NP40, and cracking condition is: 56 DEG C, 1h; 95 DEG C, 10min;
2. DNA sequencing qualification embryonic gene type knocks out situation: extract DNA, carry out PCR, electroresis appraisal, and carry out DNA sequencing, obtain genotype identification result;
A, design PCR primer are 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×Taqplus12.5ul
ddH
2O9.5ul；
C, PCR reaction conditions:
95 DEG C of denaturation 7min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 40s; 30 circulations; 72 DEG C extend 5min;
3. PCR primer checks order, there is bimodal situation in the target practice location proximate that sequencing result designs at PHEX gene primer, select bimodal sample PCR again, product glue carries out connection PGM-T carrier after reclaiming, after transforming, picking positive colony checks order again, base occurs in sequencing result near PHEX gene target site insert or base deletion, causing frame shift mutation, is then gene knockout.
The present invention is through correlation detection, success obtains phosphopenic rickets model, the acquisition of this model can the pathologic process of simulating human disease effectively, more effectively can predict the effect in clinical application such as novel vaccine, new drug and new diagnostic reagent, greatly reduce the risk of new drug development, for clinical study provides basic model simultaneously.
Accompanying drawing explanation
Fig. 1 is the structural representation of expression vector PUC57-sgRNA of the present invention;
Fig. 2 is the electrophorogram of PCR primer of the present invention qualification embryo PHEX gene knockout situation;
Wherein M:Marker
for DNA molecular normal content; 1-14: 14 embryo DNAPCR results after microinjection.15: positive control (fetal tissues); 16: water contrasts;
The primers designed of the PHEX gene of design is 448bp
Can obtain from DNA sequencing result and PCR primer electrophoresis result: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 embryos all occur different to knock out situation;
Fig. 3 be the newborn rabbit that obtains after microinjection after qualification, by control group with knock out group and record respectively and add up its situation that body is long in process of growth;
This figure is normal group and knocks out group rabbit growth curve, can find out that knock out group occurs growth retardation phenomenon in growth and development process from figure;
Fig. 4 be the newborn rabbit that obtains after microinjection after qualification, by control group with knock out group and record respectively and the situation of adding up its body weight in process of growth;
This figure is normal group and knocks out group rabbit height curve, can find out that knocking out group height is starkly lower than normal group, and growth retardation;
Fig. 5 obtains young rabbit anteserum qualification result after microinjection: content of alkaline phosphatase contrasts; This figure shows: normal group with knock out group and compare, the content knocking out group alkaline phosphatase significantly raises;
Fig. 6 obtains young rabbit anteserum qualification result after microinjection: phosphorus content contrasts; This figure shows: normal group with knock out group and compare, knock out group phosphorus content and significantly lower.
Embodiment
The present invention sets up PHEX gene knockout phosphopenic rickets model:
Step is as follows:
1) structure of CRISPR-CAS9 system sgRNA design and expression vector.
According to Dr.FengZhang(http: //crispr.mit.edu/) design and design 2 sgRNA sequence action target spots at PHEX gene the 1st exon place, synthesis two is right
Oligonucleotide chain (sgRNA1:TAGGCAATTCGAGTGCCTCTGG and AAACCCAGAGGCACTCGAATTG; SgRNA2:TAGGCTTGGCACGATCCTCTTTC and AAACGAAAGAGGATCGTGCCAAG) for the preparation of sgRNA.The oligonucleotide chain selection principle of this sgRNA: choose the oligonucleotide chain that the beginning of mark the highest and base sequence is GG.The oligonucleotide of synthesis is through annealing (being naturally down to room temperature after 95 DEG C of 5min), be connected into and cut through reclaiming PUC57-sgRNA expression vector through Bbs I enzyme, complete sgRNA vector construction, connected correct by sequence verification fragment, cloning, extracting plasmid after enlarged culturing for preparing in-vitro transcription template.
Enzyme cuts system: plasmid PUC5720 μ l
10×buffer20μl
BbsⅠ1μl
ddH
2O159μl
Enzyme cuts 37 DEG C of 3h, and after electrophoresis runs glue, use common DNA agarose gel to reclaim test kit (being purchased from Tian Gen company, Beijing, China) and reclaim, concrete operations by specification carries out.
CAS9 expression plasmid (Addgene, laboratory is bought), through linearization for enzyme restriction, after phenol chloroform purifying, is dissolved in as template in the water of nuclease free, for in-vitro transcription.The synthesis of CAS9mRNA is by test kit RNeasyMiniKit (Qiagen, No.74104) act on t7 rna polymerase in vitro to have come, the external synthesis of sgRNA utilizes t7 rna polymerase to complete by test kit MiRNeasyMiniKit (Qiasgen, No.217004) in vitro.
Enzyme cuts system: Not I 4 μ l
CAS950μl
BSA30μl
Triton30μl
10×H30μl
ddH
2O156μl
Enzyme cuts 37 DEG C of 3h, and after electrophoresis runs glue, use common DNA agarose gel to reclaim test kit (being purchased from Tian Gen company, Beijing, China) and reclaim, concrete operations by specification carries out.
2) acquisition of zygote and microinjection.
Injection follicular stimulating hormone (FSH), inject human chorionic gonadotrophin (HCG) (being purchased from Ningbo second hormone factory) afterwards, obtain zygote, by microinjection instrument, pre-mixed CAS9mRNA/sgRNA mixture is expelled to (CAS9mRNA final concentration is 150ng/ μ l, and sgRNA final concentration is 30ng/ μ l) in tenuigenin.
3) vitro culture of zygote and growth.
The zygote of microinjection being transferred in nutrient solution, is placed in 37 DEG C of constant incubators and cultivates, when growing to morula period, single embryo being transferred in centrifuge tube, with testing later with suction ovum pin.
(2) embryo PHEX gene knockout situation qualification
1) embryo's cracking
Embryo's lytic reagent is NP40, and cracking condition is: 56 DEG C of 1h
95℃10min
2) DNA sequencing qualification embryonic gene type knocks out situation.
Extract DNA, extracting method extracts test kit specification sheets according to tissue gene group to carry out operating (being purchased from Tian Gen company, Beijing, China), carries out PCR, electroresis appraisal, and carries out DNA sequencing, obtain genotype identification result.
design PCR primer is as follows:
Upstream primer: GTCAACGCTTAACAGACTC
Downstream primer: CACCTCCAACCACTTAATAC
pCR reaction system is as follows:
Template DNA 1ul
Upstream primer 1ul
Downstream primer 1ul
2×Taqplus12.5ul
ddH
2O9.5ul
pCR reaction conditions:
95 DEG C of denaturation 7min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 40s; 30 circulations; 72 DEG C extend 5min.
PCR primer checks order, if the target practice location proximate that sequencing result designs at PHEX gene primer occurs bimodal situation, then for practice shooting successfully.Select bimodal sample PCR again, product glue carries out connection PGM-T carrier after reclaiming, and after transforming, picking positive colony checks order again, base occurs in sequencing result near PHEX gene target site and inserts or base deletion, cause frame shift mutation, be then judged as gene knockout.
Below technical scheme of the present invention is clearly and completely described:
(1) set up rabbit PHEX and knock out phosphopenic rickets model.
Step is as follows :-
1) structure of CRISPR-CAS9 system sgRNA design and expression vector.
At PHEX gene, the 1st exon place designs 2 sgRNA sequence action target spots, and synthesis two is right
Oligonucleotide chain (sgRNA1:TAGGCAATTCGAGTGCCTCTGG and AAACCCAGAGGCACTCGAATTG; SgRNA2:TAGGCTTGGCACGATCCTCTTTC and AAACGAAAGAGGATCGTGCCAAG) for the preparation of sgRNA.The oligonucleotide chain selection principle of this sgRNA: choose the oligonucleotide chain that the beginning of mark the highest and base sequence is G.The oligonucleotide of synthesis is through annealing (being naturally down to room temperature after 95 DEG C of 5min), be connected into and cut through reclaiming PUC57-sgRNA expression vector through Bbs I enzyme, complete sgRNA vector construction, connected correct by sequence verification fragment, cloning, extracting plasmid after enlarged culturing for preparing in-vitro transcription template.
Enzyme cuts system: plasmid PUC5720 μ l
10×buffer20μl
BbsⅠ1μl
ddH
2O159μl
Enzyme cuts 37 DEG C of 3h, and after electrophoresis runs glue, use common DNA agarose gel to reclaim test kit (being purchased from Tian Gen company, Beijing, China) and reclaim, concrete operations by specification carries out.
CAS9 expression plasmid (Addgene, laboratory is bought), through linearization for enzyme restriction, after phenol chloroform purifying, is dissolved in as template in the water of nuclease free, for in-vitro transcription.The synthesis of CAS9mRNA is by test kit RNeasyMiniKit (Qiagen, No.74104) act on t7 rna polymerase in vitro to have come, the external synthesis of sgRNA utilizes t7 rna polymerase to complete by test kit MiRNeasyMiniKit (Qiasgen, No.217004) in vitro.
Enzyme cuts system: Not I 4 μ l
CAS950μl
BSA30μl
Triton30μl
10×H30μl
ddH
2O156μl
Enzyme cuts 37 DEG C of 3h, and after electrophoresis runs glue, use common DNA agarose gel to reclaim test kit (being purchased from Tian Gen company, Beijing, China) and reclaim, concrete operations by specification carries out.
2) acquisition of zygote and microinjection.
Injection follicular stimulating hormone (FSH), inject human chorionic gonadotrophin (HCG) (being purchased from Ningbo second hormone factory) afterwards, obtain zygote, by microinjection instrument, pre-mixed CAS9mRNA/sgRNA mixture is expelled to (CAS9mRNA final concentration is 150ng/ μ l, and sgRNA final concentration is 30ng/ μ l) in tenuigenin.
3) zygote transplation after injection and animal are cultivated.
After microinjection, by zygote transplation, carry out fetal development, carry out standardization and raise.
(2) rabbit phosphopenic rickets model phenotypic evaluation and gene type assay.
1) rabbit phenotypic results statistics.
Within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, rabbit outward appearance photo acquisition is carried out respectively at after birth.
2) genotype of DNA sequencing qualification rabbit phosphopenic rickets model.
Extract tissue DNA, extracting method extracts test kit specification sheets according to tissue gene group to carry out operating (sky root, Beijing, China), carries out PCR, electroresis appraisal, and carries out DNA sequencing, obtain genotype identification result.
design PCR primer is as follows:
Upstream primer: GTCAACGCTTAACAGACTC
Downstream primer: CACCTCCAACCACTTAATAC
pCR reaction system is as follows:
Template DNA 1ul
Upstream primer 1ul
Downstream primer 1ul
2×Taqplus12.5ul
ddH
2O9.5ul
pCR reaction conditions:
95 DEG C of denaturation 7min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 40s; 30 circulations; 72 DEG C extend 5min.
PCR primer checks order, if there is bimodal situation in the target practice location proximate that sequencing result designs at PHEX gene primer, then and may for practice shooting successfully.Select bimodal sample PCR again, product glue carries out connection carrier T after reclaiming, and after transforming, picking positive colony checks order again, inserts or base deletion if there is base in sequencing result near PHEX gene target site, cause frame shift mutation, then can be judged as gene knockout.
3) fluorescence quantifying PCR method is adopted to detect at the enterprising line correlation of mRNA level in-site.
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: by the frozen renal tissue in-80 DEG C of rabbits, uses liquid nitrogen to grind, uses TRNzol-A
+total RNA extraction reagent box extracts, and operates and carries out (being purchased from Tian Gen company, Beijing, China) according to test kit specification sheets.
reverse transcription: the RNA upper step extracted carries out reverse transcription, adopt FastQuantcDNA first chain synthetic agent box (sky root, Beijing, China), operation is carried out to specifications.
adopt BioEasySYBRGreenI Real-Time PCR Kit to carry out quantitative analysis, the present invention chooses the genes such as FGF23, SOST, PTH and carries out quantitative analysis, and reference gene is GAPDH.
4) Western blotting (WesternBlot) method is adopted to carry out the analysis of PHEX genes protein level.
WesternBlot concrete steps are as follows:
preparation of samples, first gets 10ug protein sample, adds sample-loading buffer, and boiling water sex change 5 minutes, is put on ice, 12000rpm immediately, and 4 DEG C centrifugal 5 minutes.
electrophoresis: protein sample is loaded in the sample well of the poly amic acid gel prepared, carries out electrophoresis.
electricity turns: shift " sandwich " according to the sequentially built of sponge pad, filter paper, film, gel, filter paper, sponge pad, put into electric turn trough, and 100V voltage electricity turns 3 hours.
wash film: take out film, be put in TBST and wash 15 minutes, wash 3 times continuously.
close: film is put in the TBST solution of 5% skimming milk and closes 2 hours.
primary antibodie is hatched: carry out primary antibodie dilution, 4 DEG C of overnight incubation with the TBST containing 5% skimming milk.
wash film: film TBST is washed 15 minutes, continuous 3 times.
two resist: carry out two anti-dilutions with the TBST containing 5% skimming milk, hatch 2 hours.
wash film: film TBST is washed 15 minutes, continuous 3 times.
development: need to join developing solution, uses visualizer to develop.
(3) rabbit phosphopenic rickets model phenotypic evaluation and gene type assay.
1) rabbit body length and body weight results acquisition.
Normal rabbit and the body length and the body weight that knock out rabbit is measured respectively at after birth 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks.
2) rabbit related serological result measures.
Carry out determination of serology, separation of serum respectively at 7 weeks, 14 weeks, adopt ELISA kit to measure in serum the biochemical indicators such as phosphorus, alkaline phosphatase, calcium.
Separation of serum method: get fresh blood, is put in the centrifuge tube of 1.5ml sterilizing, and 37 DEG C leave standstill 30 minutes, and centrifugal 10 minutes of 3000rpm, is drawn onto faint yellow supernatant in centrifuge tube, is put in-80 DEG C of preservations, for experimental study.
3) observe the vitals such as rabbit bone, heart, liver, spleen, kidney, lungs or organize whether pathology occurs.
Knock out rabbit in process of growth, occur dead individuality, each organ disease situation of anatomic observation, fixing organization, does tissue pathological slice.
4) rabbit skeletal image Epidemiological Analysis.
At the 7th week, get and reject clean appendicular skeleton, carry out X-ray, obtain appendicular skeleton iconography photo, carry out accordingly result analysis.
1, the sequence of PHEX gene First Exon:
ATGGAAGCAGAAACAGGGAGCAATGCGGGAACTGGAAAGACGGCCACCAGAGGCAC
TCGAATTGCCCTGGTCGTATTCATCGGCGGCACTCTCGTGCTTGGCACGATCCTCTTTCT
GG
2, fetal tissues PCR primer carries out the result (length is 448bp) of DNA sequencing:
GTCAACGCTTAACAGACTCTTGAGAGCAGCCACCAGACCACGAAAAGTGACTAAGATTCTTCTCGTGTGCTCTCTACGGC
CCTTCTGATGGAAGCAGAAACAGGGAGCAATGCGGGAACTGGAAAGACGGCCACCAGAGGCACTCGAATTGCCCTGGTCG
TATTCATCGGCGGCACTCTCGTGCTTGGCACGATCCTCTTTCTGGGTAAGTGGAGCCCTGGAGGCCGGGGGCACGGGGCA
TGTGCTGAGAAATCCCTTGTGCTTTATTCTAGCCTTGTCATAATGCAAGTGTGCGACTGTTTGCTTGTGTTAGAAGGTGA
TGCAGTCTTGGTTTCTATCAAATGGGCAAATGAAAAAGGCTTTGCTTTCAGAGGCTACATGCATAAATACAGTCAAGAAC
TGAATATGTACTGCAGAGGGCAAAGTGGGTATTAAGTGGTTGGAGGT
3, PUC57-sgRNA1 sequence (italic underscore is labeled as sgRNA1 oligonucleotide chain):
CAGTGATTGGAGATCGGTACTTCGCGAATGCGTCGAGATATTGGGTCTTTAAAAGCACCGACTCGGTGCCACTTTTTCAA
GTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTA
AAACCCAGAGGCACTCGAATTG CCTATAGTGAGT
CGTATTAATTGGGTATCGGATGCCGGGACCGACGAGTGCAGAGGCGTGCAAGCGAGCTTGGCGTAATCATGGTCATAGCT
GTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTG
CCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTG
CATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCT
GCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATA
ACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAT
AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATA
CCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTC
TCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG
GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGGTAACTATCGTCTTGAGTCCAACCCGGTAA
GACACGACTTATCGCCACTGGCAGCAGCCACTGGTACAGGATTAGCAGAGCGAGTATGTAAGCGTTGCTACAGAGTTCTT
GAAGTGGTGCCTAACTACGGGCTTACCACTAAGGA
4, PUC57-sgRNA2 sequence (italic underscore is labeled as sgRNA2 oligonucleotide chain):
CGGCAGTGATTGGAGATCGGTACTTCGCGAATGCGTCGAGATATTGGGTCTTTAAAAGCACCGACTCGGTGCCACTTTTT
CAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTA
AAACGAAAGAGGATCGTGCCAAG CCTATAGT
GAGTCGTATTAATTGGGTATCGGATGCCGGGACCGACGAGTGCAGAGGCGTGCAAGCGAGCTTGGCGTAATCATGGTCAT
AGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGG
GGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCA
GCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACT
CGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGG
GATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT
CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA
GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCC
TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAA
GCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGG
TAAGACACGACTTATCGCACTGGCAGCAGCACTGTAACAGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT
Claims (3)
1. employing knocks out the method that technology sets up phosphopenic rickets model, it is characterized in that:
1. the structure of sgRNA expression vector: the 1st exon place designs 2 sgRNA sequence action target spots at PHEX gene, synthesize two pairs of oligonucleotide chains and prepare sgRNA, wherein two pairs of oligonucleotides are:
SgRNA1:TAGGCAATTCGAGTGCCTCTGG and AAACCCAGAGGCACTCGAATTG;
SgRNA2:TAGGCTTGGCACGATCCTCTTTC and AAACGAAAGAGGATCGTGCCAAG;
The oligonucleotide of synthesis is through annealing, and annealing conditions is: be naturally down to room temperature after 95 DEG C of 5min, is connected into and cuts through reclaiming PUC57-sgRNA expression vector through Bbs I enzyme, complete sgRNA vector construction;
Wherein: enzyme cuts system: plasmid PUC5720 μ l
10×buffer20μl
BbsⅠ1μl
ddH
2O159μl
Enzyme cuts 37 DEG C of 3h, after electrophoresis runs glue, uses common DNA agarose gel to reclaim test kit and reclaims;
2. the synthesis of CAS9mRNA: CAS9 expression plasmid, through linearization for enzyme restriction, after phenol chloroform purifying, is dissolved in the water of nuclease free;
Enzyme cuts system: Not I 4 μ l
CAS950μl
BSA30μl
Triton30μl
10×H30μl
ddH
2O156μl
Enzyme cuts 37 DEG C of 3h, after electrophoresis runs glue, uses common DNA agarose gel to reclaim test kit and reclaims;
3. the acquisition of zygote and microinjection: injection follicular stimulating hormone, inject human chorionic gonadotrophin afterwards, obtain zygote, by microinjection instrument, pre-mixed CAS9mRNA/sgRNA mixture is expelled in tenuigenin, wherein CAS9mRNA concentration is 150ng/ μ l, and sgRNA concentration is 30ng/ μ l;
4. the cultivation of zygote and growth: transfer in nutrient solution by the zygote of microinjection, be placed in 37 DEG C of constant incubators and cultivate, when growing to morula period, transfers to single embryo in centrifuge tube with suction ovum pin.
2. employing according to claim 1 knocks out the method that technology sets up phosphopenic rickets model, it is characterized in that: the oligonucleotide chain selection principle of sgRNA: choose the oligonucleotide chain that the beginning of mark the highest and base sequence is GG.
3. employing according to claim 1 knocks out the method that technology sets up phosphopenic rickets model, it is characterized in that: embryo PHEX gene knockout situation authentication method:
1. embryo's cracking: embryo's lytic reagent is NP40, and cracking condition is: 56 DEG C, 1h; 95 DEG C, 10min;
2. DNA sequencing qualification embryonic gene type knocks out situation: extract DNA, carry out PCR, electroresis appraisal, and carry out DNA sequencing, obtain genotype identification result;
A, design PCR primer are 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×Taqplus12.5ul
ddH
2O9.5ul；
C, PCR reaction conditions:
95 DEG C of denaturation 7min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 40s; 30 circulations; 72 DEG C extend 5min;
3. PCR primer checks order, there is bimodal situation in the target practice location proximate that sequencing result designs at PHEX gene primer, select bimodal sample PCR again, product glue carries out connection PGM-T carrier after reclaiming, after transforming, picking positive colony checks order again, base occurs in sequencing result near PHEX gene target site insert or base deletion, causing frame shift mutation, is then gene knockout.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450727A (en) * | 2014-11-13 | 2015-03-25 | 深圳华大基因科技有限公司 | Pathogenic gene for X-linked hypophosphatemic rickets as well as protein encoded by pathogenic gene and application of pathogenic gene |
-
2015
- 2015-09-06 CN CN201510557129.7A patent/CN105400810B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450727A (en) * | 2014-11-13 | 2015-03-25 | 深圳华大基因科技有限公司 | Pathogenic gene for X-linked hypophosphatemic rickets as well as protein encoded by pathogenic gene and application of pathogenic gene |
Non-Patent Citations (2)
| Title |
|---|
| 董训兰等: "大剂量维生素D对佝偻病模型的实验观察", 《蚌埠医学院学报》 * |
| 赵琳等: "佝偻病大鼠模型建立及骨密度变化的实验研究", 《昆明医科大学学报》 * |
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