CN109207524A

CN109207524A - The foundation and application of human obesity's zebra fish model based on FTO gene

Info

Publication number: CN109207524A
Application number: CN201710545564.7A
Authority: CN
Inventors: 赵庆顺; 董晓华; 储乐乐; 池军
Original assignee: Nanjing Yao Yu Biotechnology Co Ltd
Current assignee: Nanjing Yao Yu Biotechnology Co Ltd
Priority date: 2017-06-29
Filing date: 2017-06-29
Publication date: 2019-01-15

Abstract

The present invention relates to the foundation and application of human obesity's zebra fish model based on FTO gene, specific steps include the exon design CRISPR sequence for zebra fish fto gene encoded key functional domain, screening confirmation can effectively guide the active sgRNA of Cas9 cutting target gene, then active sgRNA is used, prepare the first person of building of fto gene knockout, sexal maturity is grown to just not build person, enables its pangamy, generates F1 generation；By the genotype identification to F1 individual, the zebra fish mutant of fto gene knockout is screened；Pass through the analysis to fto homozygous mutation, the afunction of establishment fto is beneficial to the reduction of the accumulation of fat of zebra fish prelarva, the zebra fish model of the human obesity based on fto gene is established, and then fto can be applied to screening as mark gene and establish the novel drugs and new method for the treatment of human obesity.

Description

The foundation and application of human obesity's zebra fish model based on FTO gene

Technical field

The invention belongs to field of biotechnology, and being related to screening determination can carry out genome editor's to zebra fish fto gene The preferred embodiment that sgRNA sequence, preparation zebra fish to gene target are mutated, and using zebra fish fto as significant gene The application of the zebra fish model of screening treatment human obesity disease drug.

Technical background

Obesity is that fat accumulation extra in vivo is more than to cause the energetic supersession of puzzlement unbalance human health to a certain degree Chronic disease.People use BMI (body mass index, body mass index) usually to judge whether individual is fat.Because body fat increases Adding makes weight be above standard weight 20% or body mass index [BMI=weight (Kg)/(height)²(m²)] it is known as obesity greater than 24 Disease.Obesity will increase the illness probability of Other diseases, especially heart disease, type-2 diabetes mellitus, obstructive sleep respiratory distress, cancer The diseases such as disease and osteoarthritis.In recent decades, obesity patient increases at an amazing speed in the world.Especially Virgin, Adolescent Obesity increases increasingly swift and violent (1,2).In 2014, there are about 6,000,000 adults (13%) and forty-two million 5 years old or less Children suffer from obesity.Obesity is considered as 21 century unhealthful disease the most serious.

The fat cause of disease is broadly divided into external cause and internal cause, and external cause is with hyperphagia, and based on Lack of Movement, caloric intake is extra Heat consumption increases Fatty synthesis by (3,4).And internal cause is if due to caused by genetic predisposition.Similar to other many Disease, obesity are the results to interact between inherent cause and environmental factor.The morbidity of the simple obesity of the mankind has centainly Genetic background.Polymorphism existing for many genes controls appetite and metabolism to be easier to make body in the presence of enough energy Increase to be liable to obesity again.Show the common bariatric patient of 40-70% in family and twinborn research It is fat (5,6) to belong to heredity.By the end of 2006, it has been found that on human genome with obesity-related polymorphic site Already exceed 41.2008, the mankind filtered out first gene FTO (fat mass that can lead to human obesity disease and obesity associated gene).Loos discovery, FTO is an obesity-prone gene, and FTO gene first includes Some mutation of son are positively correlated with obesity.The study found that FTO gene First Intron carries the homozygous crowd's of obesity SNP Weight is 3-4kg averagely heavier than the crowd for not carrying SNP, and the risk for suffering from obesity will increase by 1.67 times (7).Other one About showing that FTO existing some SNP sites in First Intron are close with type-2 diabetes mellitus disease in the report of FTO (8) are closed in cut phase.But by control patient BMI index, it is found that the diabetic phenotype of these patients disappears, this explanation, the mutation of FTO It is to influence secondary diabetes conditions by influencing weight.These are statistics indicate that the mutation of FTO gene is close with obesity Cut phase is closed.

In molecular biology level, in mouse, xenopous laevis, zebra fish, drosophila and nematode isotype animal is raw Object event and people enjoy identical molecular mechanism.Therefore, the effective in body mould of human diseases can be made using model animal Type.In all model animals, zebra fish is the bridge connected between invertebrate and mammalian animal model, in human gene 87% can find homologous gene wherein.It selects to move using zebra fish as mode currently, the whole world has more than 1000 laboratories Object carries out the research from the different research fields such as fundamental biological knowledge, Environmental Toxicological Assessment to medicament research and development, and the country has more than 100 Laboratory is using zebra fish as main study subject.Zebra fish raising is simple, and feeding environment control is more in certain egg laying amount, and every time one It can lay eggs 200-300 pieces weekly to fish, embryonic development is transparent, and growth cycle is shorter, is to carry out embryonic development mechanism, genome The good material of research and pathogenic mechanism research.For the tumor susceptibility gene FTO closely related with human obesity, people's FTO base Because being located at No. 16 chromosome, FTO albumen is made of 505 amino acid；And in zebra fish, with people's FTO homologous gene fto In No. 7 chromosomes of zebra fish, albumen is made of 579 amino acid.Bioinformatic analysis discovery, fto gene and people's FTO base Because of ortholog (there are co-linear relationships with aktip and AKTIP gene respectively).In its expression product protein Amino acid composition on, it is found that the phase same sex (identity) of FTO and fto is high, two functional domains (are FTO catalysis respectively Domain and FTO C- terminal domain) the phase same sex is higher, and further support FTO gene highly conserved in people and zebra fish.It is based on The above reason, we use zebra fish for model, using genome editing technique, targeting mutation fto gene.Mutant is divided Analysis discovery, the homozygous mutation of fto cause fat cell to substantially reduce in the intracorporal accumulation of zebra fish.The result shows that fto is one With obesity-related gene, the mark gene of screening treatment obesity may be used as.

Bibliography:

1.Weiss R, Dziura J, Burgert TS, Tamborlane WV, Taksali SE, et al., Obesity And the metabolic syndrome in children and adolescents.N Engl J Med.2004,350 (23):

2.Fleisch AF, Agarwal N, Roberts MD, Han JC, Theim KR, et al., Influence of serum leptin on weight and body fat growth in children at high risk for adult Obesity.J Clin Endocrinol Metab.2007,92 (3): 948-954.

3.Abelson P, Kennedy D.The obesity epidemic.Science 2004；304:1413.

4.Keith SW, Redden DT, Katzmarzyk PT, Boggiano MM, Hanlon EC, et al., Putative contributors to the secular increase in obesity:exploring the roads less traveled.Int J Obes 2006；30:1585-1594.

5.Maes HH, Neale MC, Eaves LJ.Genetic and environmental factors in relative body weight and human obesity. Behav Genet 1997；27:325-351.

6.Clement K, Sorensen TIA.Obesity:Genomics and Postgenomic.Informa Healthcare:New York, 2008.

7.Loos, R.J., and Bouchard, C.FTO:the first gene contributing to common Forms of human obesity.Obesity reviews:an official journal of the International Association for the Study of Obesity 2008；9,246-250.

8.Frayling TM, Timpson NJ, Weedon MN, Zeggini E, Freathy RM, et al., A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity.Science 2007；316:889-894.

Summary of the invention

For the zebra fish model for establishing human obesity, we are struck using the targeting of CRISPR/Cas9 genome editing technique Except fto gene, the influence that obesity of the fto gene pairs characterized by fat accumulation occurs is confirmed.CRISPR/Cas9 technology is It is no successfully to edit fto gene, realize the gene knockout of zebra fish fto, key, which is whether to screen, effectively to be guided Cas9 identifies the sgRNA of target gene sequence (CRISPR sequence).When sgRNA can effectively guide Cas9 targets identification target base Cause, Cas9 therein can cut off 3 ' -5 ' phosphodiester bonds between 3 to 4 nucleotide before the PAM of CRISPR sequence, from And form the double-strand break (DSB) of genomic DNA.Once DSB is formed, embryonic cell will start non-homologous end joining reparation or Micro- homologous end connects repair mechanism, as a result the DSB of revision points group introduces insertion and deletion (Indel) mutation at DSB.It is logical It crosses and screens the mutant for carrying and having frameshift mutation gene in F1 generation, so that it may the zebra fish mutant for obtaining fto gene knockout, from And it is expected to understand effect of the fto in obesity generation.To realize this target, we, will according to the analysis of the functional domain of Fto CRIPSR sequence design is on the 3rd exon corresponding to first functional domain.We devise 8 CRISPR sequence (tables altogether One).According to above-mentioned CRISPR sequence, we are prepared for corresponding sgRNA (sgRNA1 to sgRNA8).With microinjection skill Art, 8 sgRNA of acquisition are divided into two groups, and (sgRNA1, sgRNA3, sgRNA5 and sgRNA7 are group 1；sgRNA2,sgRNA4, SgRNA6 and sgRNA8 is 2) group, into zebra fish fertilized egg, carries out the sgRNA with Cas9 albumen co-injection respectively Activity verifying.Cas9 can be effectively guided to identify target as a result, present invention firstly provides two according to activity verifying The sgRNA1 and sgRNA3 of CRISPR sequence.

SgRNA1 and sgRNA3 and Cas9mRNA are injected into zebra fish fertilized egg (F0), embryonic development to be injected to property at When ripe, its pangamy is enabled, obtains F1 individual, by clip tail fin, genotype identification is carried out to F1 individual.It reflects according to genotype It is fixed as a result, invention further provides the preparation methods for the zebra fish mutant for carrying fto knock out mutants.

When F1 heterozygote grows to sexal maturity, F1 is selfed, obtains F2 for embryo.To embryonic development to after fertilization 4 days (4dpf) carries out staining analysis to embryo with oil red, according to analysis result, it has been found that when zebra fish fto afunction, The fat accumulation amount of embryo is remarkably decreased.The present invention provides human obesity's mould based on zebra fish fto gene again as a result, Type, zebra fish fto can be used as the marker gene of screening obesity treating medicine, can make any substance of fto expression decline It will be provided with inhibiting the effect of obesity.

Detailed description of the invention

The conserved structure function domain analysis of Fig. 1 zebra fish Fto protein.Fto includes two conserved functional domains, respectively FTO_NTD superfamily and FTO_CTD superfamily.

Fig. 2 can effectively guide the sgRNA activity verifying analysis of Cas9 targeting cutting fto gene.Utilize VectorNTI software (totally 10 recons) is compared to recon sequencing sequence, shows that sgRNA1 and sgRNA3 are active, and sgRNA1 Activity rate is that 20%, sgRNA3 activity rate is 10%.

The verifying analysis of Fig. 3 sgRNA2/4/6/8 activity.Recon sequencing sequence is compared using Vector NTI software SgRNA2, sgRNA4, sgRNA6, sgRNA8 are inactive or active lower to be shown to analysis (totally 10 recons).

The sequencing peak figure of the fto mutation allele partial sequence of Fig. 4 zebra fish F1 generation mutant 3 carryings

The truncated protein structural representation of the fto mutation allele predictive coding of Fig. 5 zebra fish F1 generation mutant 3 carryings Figure.WT: wild type Fto protein structure schematic diagram, MUT1: the protein of one of fto mutation allele predictive coding Structural schematic diagram, the protein structure schematic diagram of MUT2: the second fto mutation allele predictive coding.

The sequencing peak figure of the fto mutation allele partial sequence of Fig. 6 zebra fish F1 generation mutant 11 carryings.

The truncated protein structure of the fto mutation allele predictive coding of Fig. 7 zebra fish F1 generation mutant 11 carryings is shown It is intended to.WT: wild type Fto protein structure schematic diagram, MUT1: the egg of predictive coding after one of fto allelic mutation White matter structural schematic diagram.

The sequencing peak figure of the fto mutation allele partial sequence of Fig. 8 zebra fish F1 generation mutant 13 carryings.

The truncated protein structure of the fto mutation allele predictive coding of Fig. 9 zebra fish F1 generation mutant 13 carryings is shown It is intended to.Wild type Fto protein structure schematic diagram, MUT1: the protein of predictive coding after one of fto allelic mutation Structural schematic diagram.

The zebra fish mutant prelarva inner lipid storage capacity that Figure 10 carries fto homozygous mutation is reduced.Oil red O is to development to 4 It zebra fish prelarva is dyed, and finds compared with wild-type zebrafish, fto mutant zebra fish prelarva body fat contains Amount significantly reduces.

Specific embodiment

Experimental method in embodiment is unless otherwise specified conventional method.

The screening of 1 activity sgRNA of embodiment

(1) design of the CRISPR of targeting mutation zebra fish fto gene

1, gene information is analyzed

The CDS of 1.1 coding zebra fish fto genes

ATGAAACCGAGGCAGCGTAAACAGTACTTCAGGAACATGAAAAGATCGGATGACAGTGAGAGGGAAAAGAGGAGAA AGAGGCGAAGGCTTCTACAGGAACTAGGAGAGCAGAGGATTCCATATCTTTCCCCAACAGACCCAGGATTTCAGGA CCTGTGGGACTCCAGTTATGCGGGCCTGGCTTTGCGGCAGTCTGGTACCCTTCCAGAAGGTCTCCATGAGAAGGTT CAATCTGCTCTGTTAACTCTACAGCGGCATGGATGTCTCCTCCGAGATCTTGTCCGAGTTCGAGACCGGGACGTCT TCACGGCCGTTTCACGCGCTCTCGTGGGTCAGCCGGGCTACACGTACCGTTATCTGGACACTCGTCTGTTCACCAT CCCCTGGCACTGTGAAGGAGAAGAGGGCCAGAAAGATGAGAAAGGCAAACCTTGCTGTGACTCCGACCTGAGGGAC GCTTGTAAAGCATTATGGGAGCTCAATCAGTTCTTTTGCTCGGATGTAAAGCAGCAGACAAATGCAAGAGGTGTCA AGCGTACCCGCAGTGACACTGAAAACAGCGAAGATGCGCCAGGGGAGGGAATGTGCGAAGAAGAGAGTGTTAAGGA CAGGTTAGTTGAGGAGAAAACCATAGAGGAGGAGGAGGAAGACAGTGGACAGGGCTGCTCTCATTCCTCACCTCCT TCATCCACTCCGCCAGCTGCTCAGGCCAGCCTGGTTCAGTTTAACGTCACGCTAATCAACTACATGAACCCTGCAG CCATGACCCAGCTGAAGGAAGAGCCATATTACGGCATGGGCAAAATGGCAGTCGGTTGGCATCATGATGAGAACCT GGTTCCTCTCTCACCAGTTGCCGTTTACAGCTATAGCTGCCCAGCAGAGCCAAAGAATGAAGGAGTTACTGAGAAA GACGGGGAGGGAAAGAGCAAAGAAAAAGAAAGAGAGGCCAAAGGTGAAGGAACAAGTACAGAAGAAGTGAAGAAAG AAGGAGAAGCTTCGGTGAAGGAGGACGTGGAAAAAGAGAAGACGTGTTGGCGAGTTGGTCTGAAGGTGGCCTGGGA CATCCACACACCAGGTCTGGCTTTGCCTCTCCAATCTGGAGACTGCTACTATATGACAGATGATCTGAACCGGACA CATCAGCACTGTGTACTGGCTGGTGACACAGCTAGATTCAGCTCAACTCACAGAGTCGCACAGTGTTGTACGGGTA CACTGGACTACATACAGAAACGTTGCTCTGAGGCCTTGGAGAATTTACACAGCGACCCCGAGAAGAACGCCAAGAG CCTGATTTCCCTCCTGCCCTCCATCCTCCAACGCATTGAGGATATCCACAACGAGGTGGAGTTTGAGTGGCTGAGG CAGTACTGGTTCCAGGGCCGGCGTTACGCTCGATTCTGCAGCTGGTGGACTAAACCCATGGAGCAGATGGAGAAAG ATTGGAAGGAAATGGAGAGAATGACTCAGCTGCTGTTGGTGGTGGTTGAGGATGAGGCCACGGCACAGGAGAACAG AAGAGAGATGGCAGACGTGTTACTAAATGCGCTGACCGACAGACAGCAGCACAGACAGACATGGAGAGACAGATGC CAGTCCAGCCTGGCGCAGACTTTGCCCCCTGAGGAAGCACCTGTGGACAGACCCTATTGGAGCAATGATGATCCTG ACATGCCCCTCCCCTTTGACCTCTCTGACATCATCAACCGCGTTGAGTCGCTTCTATGGAGAATGTAA

1.2 zebra fish Fto protein sequences

The conserved structure functional domain of 1.3 protein

See Fig. 1.

2, the design of CRISPR

The exon 3 sequence of 2.1 zebra fish fto genes

Yellow font is the CRISPR of design.The sequence of underscore is PAM sequence NGG (RC:CCN).

The CRISPR design of table one, targeting mutation zebra fish fto gene

(2) the active identification of sgRNA of targeting mutation zebra fish ucp1 gene

1, the genotype identification of target gene

1.1 target gene group sequences to be amplified

Yellow highlight mark is the CRISPR of design, is PAM sequence NGG (RC:CCN) with underscore, the highlighted mark of green It is denoted as primer sequence.

The design of primers of 1.2PCR amplification

Sequence names	Sequence	Purposes
			F-Fto-1	caaaaaccagtaggtctgtgga	PCR forward primer
R-Fto-1	Ggctgcagggttcatgtagt	PCR reverse primer

2, the external synthesis of sgRNA

The preparation (PCR method) of 2.1sgRNA template

2.1.1PCR primer

Forward primer sequence is as follows:

Note: the high bright part of grey is that T7 starts subdivision, and underscore part is the CRISPR sequence of customization, small letter Mother is part sgRNA skeleton template sequence.

Reverse primer (for universal primer) R-Common:AAAAAAAGCACCGACTCGGTGCCAC

2.1.2 template sequence is transcribed in vitro in the estimated sgRNA obtained

Note: the high bright part of grey is that T7 starts subdivision, and underscore part is the CRISPR sequence of customization, small letter Mother is sgRNA skeleton template sequence.

The in-vitro transcription of 2.2sgRNA

2.2.1 process is transcribed in vitro

Above-mentioned template after purification is transcribed in vitro with t7 rna polymerase.Use RNA in-vitro transcription kit (MAXIscript SP6/T7, Ambion, USA), sequentially adds 10 × Transcription by kit specification requirement Buffer 2μl、10mM ATP 1μl、10mM CTP 1μl、10mM GTP 1μl、10mM UTP 1μl、T7RNA 2 μ l of polymerase mix, 12 μ l of template DNA, after flicking mixing centrifugation, 37 DEG C of water-bath 1h.

DNase I (Ambion, USA) 1 μ l, 37 DEG C of water-bath 15min, to remove removing template is added.

30 μ l DEPC water extended volumes to 50 μ l are added later, while 5 μ l nuclease free 3M sodium acetates (pH5.2) are added With the dehydrated alcohol (domestic analysis is pure) of 3 times of volumes, -80 DEG C of precipitates overnights.4 DEG C of 12,000g are centrifuged 20min, after removing supernatant, It is deposited in draught cupboard to dry, it is spare to be stored in -80 DEG C of refrigerators after being then resuspended with 15 μ l nuclease free ultrapure waters.

2.2.2sgRNA sequence

Fto-sgRNA1:

GGACUCCAGUUAUGCGGGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGCUUUUUUU

Fto-sgRNA2:

GAGCAGAUUGAACCUUCUUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGCUUUUUUU

Fto-sgRNA3:

GAACUCGGACAAGAUCUCGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGCUUUUUUU

Fto-sgRNA4:

GGGCUACACGUACCGUUAUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGCUUUUUUU

Fto-sgRNA5:

GAGGGCCAGAAAGAUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGCUUUUUUU

Fto-sgRNA6:

GCUCAAUCAGUUCUUUUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGCUUUUUUU

Fto-sgRNA7:

GAAAACAGCGAAGAUGCGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGCUUUUUUU

Fto-sgRNA8:

GGAGGAGGAAGACAGUGGACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGCUUUUUUU

3, Cas9 albumen

The Cas9 albumen bought from commercial company (spun gold is auspicious) is diluted to 400ng/ μ l with DEPC water, be placed in after packing- It is saved in 20 DEG C of refrigerators.

4, the microinjection of zebra fish fertilized egg

Zebra fish fertilized egg is collected according to a conventional method.Above-mentioned sgRNA1, sgRNA3, sgRNA5, sgRNA7 is (final concentration of 50ng/ μ l)/sgRNA2, sgRNA4, sgRNA6, sgRNA8 (final concentration of 50ng/ μ l) and Cas9 albumen it is (final concentration of 200ng/ μ l) mixing after, micro- injection zebra fish fertilized egg, injection volume be the every embryo of 1nl.

5, the recombination confirmation of target gene editor sub (Edits)

The preparation of 5.1 target gene group fragment templates

When embryonic development to be injected is to pf for 24 hours, 5 pieces of embryos are respectively taken, " the zebra fish genotype identification for using our company to produce Kit " prepare genomic DNA template, specific reaction condition are as follows: 65 DEG C of 30min, 95 DEG C of 5min, 16 DEG C of 1min, 4 DEG C

5.2PCR amplification reaction system (20 μ l)

2 × Mastermix (Transgene), 10 μ l, the ultrapure water of 7 μ l, 1 μ l forward and reverse (F1-lgmn and R1-lgmn) The genomic DNA template of primer (5 μM) and 1 μ l prepared by 1.6.5.1.

5.3PCR reaction condition

95 DEG C of 3min, 30 × (95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min), 72 DEG C of 10min, 4 DEG C.

The recombination of 5.4 target gene pcr amplification products

The pcr amplification product of F1-fto and R1-fto is recombined into pGEM-T Easy carrier (reactions of 5 μ l total volumes), Reaction composition are as follows:

1.5 μ l pcr amplification products (being derived from 5.5.2)

2.5μl 2×Ligation Buffer

0.5 μ l T4DNA ligase

0.5 μ l pGEM-T Easy carrier (Promega)

Reaction time are as follows: incubation at room temperature 60min

The conversion of 5.5 recons

By 100 μ l bacillus coli DH 5 alpha competent cell (pfu >=10 of above-mentioned 5 μ l connection product⁸) converted.Then It is coated on the LB plate of benzyl containing ammonia (50 μ g/ml), is inverted overnight incubation at 37 DEG C.

5.6 target gene group DNA edit the recombination confirmation of sub (Edits)

According to stripe size, chooses clone and directly send sequencing, sequencing primer T7.Group 1 (sgRNA1, sgRNA3, SgRNA5, sgRNA7) survey 10 is sent, sequencing result feeds back 10.Fig. 2 is shown in particular sequence comparison.Target gene group DNA is edited Sub (Edits) sequence alignment result shows that sgRNA1, sgRNA3 are active, and sgRNA1 activity rate is 20%, sgRNA3 activity Rate is 10%.1 (sgRNA2, sgRNA4, sgRNA6, sgRNA8) of group send survey 9, and sequencing result feeds back 9.Particular sequence ratio To seeing Fig. 3.Target gene group DNA edit sub (Edits) sequence alignment result show sgRNA2, sgRNA4, sgRNA6, SgRNA8 is inactive or active is lower than 5%.

6, conclusion:

In summary experimental result can be confirmed that the sgRNA1 and sgRNA3 can effectively guide Cas9 targeting cutting mesh Mark gene.

2 zebra fish fto knock out mutants body production program of embodiment

(1) the just preparation for the person of building

By the method for 4 descriptions of scheme (two) in embodiment 1, zebra fish fertilized egg is collected according to a conventional method.It will be above-mentioned After sgRNA1 and sgRNA3 (final concentration of 50ng/ μ l) and Cas9 albumen (final concentration of 200ng/ μ l) mix, micro- injection spot Horse fish fertilized egg (injection volume is the every embryo of 1nl), prepares the person of building (Founder) at the beginning of fto knock out mutants body.

(2) screening of F1 mutant

1, prepared by F1 generation individual templet gene group DNA

Breeding and raising of the 1.1F1 for zebra fish

By the F0 embryonic feeder for having injected Cas9/sgRNA to sexal maturity, its pangamy is then enabled, F1 embryo is obtained.It will F1 embryo routinely raises to sexal maturity, carries out genotype identification, screening-gene editor's mutant.

The 1.2 noninvasive materials of tissue

The F1 adult fish at 3~4 monthly ages is taken, clip part tail fin tissue is respectively put into 200 μ l PCR pipes, then in order It is placed in and stores on ice.

1.3 genomic templates DNA preparation

The 10 μ l of YSY buffer that Nanjing Yao produces along Yu is added into the 200 μ l PCR pipes containing tail fin tissue.Quickly After centrifugation, PCR pipe is put into PCR instrument, is reacted as follows: 65 DEG C of 30min, 95 DEG C of 5min, 16 DEG C of 1min, 4 DEG C.

2, the amplification of target gene group DNA

The genome sequence of 2.1 recognition sites containing sgRNA

Red font mark is used for the primer sequence of PCR amplification, the highlighted mark sgRNA recognition site sequence of yellow.

The design of 2.2 genomic fragment amplimers

F-fto-1:CAAAAACCAGTAGGTCTGTGGA

R-fto-1:GGCTGCAGGGTTCATGTAGT (RC:ACTACATGAACCCTGCAGCC)

2.3 genomic DNA fragment PCR amplifications

2.3.1 the composition of reaction

2 × Mastermix (Vazyme), 10 μ l, the ultrapure water of 7 μ l, 1 μ l forward and reverse (F-phf8-1 and R1R-phf8- 1) the cell genomic dna template of primer (5 μM) and 1 μ l obtained by 1.3.2 step.

2.3.2PCR reaction condition

95 DEG C of 3min, 35 × (95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 50s), 72 DEG C of 10min, 4 DEG C.

3, the screening and identification of the F1 mutant of fto mutation allele is carried

It directly send the PCR product of fto mutant to sequencing, the mutation equipotential for confirming that its mutant carries is read by peak figure The genotype of gene.

The confirmation of 3.1 mutant, 3 genotype

3.1.1 3 genotype of mutant and the truncated protein sequence of the mutation allele of prediction coding

3.1.2 peak figure (part) is sequenced

See Fig. 4.

The target gene group segment pcr amplification product direct Sequencing result figure (part) of Fig. 4 mutant 3

3.1.3 the truncated protein structural schematic diagram for the mutated gene coding predicted

See Fig. 5.

The confirmation of 3.2 mutant, 11 genotype

3.2.1 11 genotype of mutant and the truncated protein sequence of the mutation allele of prediction coding

3.2.2 peak figure (part) is sequenced

See Fig. 6.

The target gene group segment pcr amplification product direct Sequencing result figure (part) of Fig. 6 mutant 11

3.2.3 the truncated protein structural schematic diagram for the mutated gene coding predicted

See Fig. 7.

The truncated protein structural schematic diagram for the mutation allele predictive coding that Fig. 7 mutant 11 carries

The confirmation of 3.3 mutant, 13 genotype

3.3.1 13 genotype of mutant and the truncated protein sequence of the mutation allele of prediction coding

3.3.2 peak figure (part) is sequenced

See Fig. 8.

The target gene group segment pcr amplification product direct Sequencing result figure (part) of Fig. 8 mutant 13

3.3.3 the truncated protein structural schematic diagram for the mutated gene coding predicted

See Fig. 9.

The truncated protein structural schematic diagram for the mutation allele predictive coding that Fig. 9 mutant 13 carries

4, conclusion: the F1 mutation zebra fish that 3 tails carry fto frameshift mutation is screened altogether.Wherein No. 11 and No. 13 zebra fish Mutation allele entrained by mutant is consistent.

3 zebra fish fto afunction of embodiment reduces the accumulation of fat

1, embryo prepares

The F1 generation heterozygote zebra fish culture for carrying fto mutation is only in Nanjing Yao Shunyu Biotechnology Co., Ltd zebra fish In vertical cultivation unit, when its sexal maturity, enables F1 generation individual freedom mate, routinely collect fertilized eggs, all embryos and juvenile fish Cultivation is at 28.5 DEG C of room temperature, for testing when embryonic development was to 4 days.

2, the preparation of oil red O

It is purchased from Sigma company, mother liquor is configured to 0.5% (w/v), and 0.5g oil red O is taken to be dissolved in 100ml isopropanol.

3, embryo fixes

Take 2g paraformaldehyde be dissolved in PBS in 65 DEG C of water-baths (76g NaCl, 9.9gNa2HPO4,3.6gNaH2PO4, It is dissolved in 10L water, pH is adjusted to 7.0), be configured to 4%PFA, for fixing 4dpf embryo.

4, oil red O stain

1) 4 days zebra fish juvenile fish are taken, 4% 4 DEG C of paraformaldehyde fixes 12 hours

2) PBST is washed 2 times

3) 0.5% oil red O room temperature dyes 4-6 hours

4) PBST is washed 3 times, and 60% isopropanol is washed 2 times, every time 5 minutes

5) PBST is washed once, is observed under anatomical lens, digital camera photographs to record.

5, genotype identification and interpretation of result

To the ucp1 zebra fish mutant through oil red O stain, genome is extracted, by the scheme write listed by embodiment 2 to every A embryo carries out genotype identification one by one.As a result, it has been found that the accumulation of embryo's fat of ucp1 homozygous mutation significantly reduces (Figure 10).

The zebra fish mutant prelarva inner lipid storage capacity that Figure 10 carries fto homozygous mutation is reduced.Oil red O is to development to 4 It zebra fish prelarva is dyed, and finds the ucp1 mutant zebra fish prelarva body fat compared with wild-type zebrafish Content significantly reduces.

Claims

1. the foundation and application of the human obesity zebra fish based on FTO gene, feature provide firstly two can be effective The active sgRNA for guiding Cas9 identification zebra fish fto gene, the zebra fish for having made fto gene knockout using active sgRNA are prominent Variant provides the zebra fish model as human obesity, thus establish zebra fish fto gene can be used as mark gene answer For screening and establishing the novel drugs and new method for the treatment of human obesity.

2. feature as described in claim 1 can effectively guide Cas9 to identify that the active sgRNA of zebra fish fto gene is respectively SgRNA1 and sgRNA3.

3. the feature as described in right 1, the mutant of zebra fish be respectively carry insertion 4bp and lack 4bp fto it is invalid etc. The heterozygote of position gene, sexually matured heterozygote selfing generate zebra fish model of the homozygote as human obesity.

4. the zebra fish model as described in right 3, the age of zebra fish prelarva is after fertilization 3 days, 4 days, 5 days, 6 days and 7 days.

5. the zebra fish model as described in right 3, applied to screening and the novel drugs and new method of establishing treatment human obesity.

6. the feature as described in right 1, mark gene of the fto gene as human obesity, homozygous mutation lead to zebra fish Prelarva Fat Accumulation is suppressed.

7. the feature as described in right 6, the novel drugs of the reduction instruction treatment human obesity of zebra fish fto gene expression dose With the screening and foundation of new method.

8. the feature as described in right 6, zebra fish fto gene can carry out various gene modifications, include but are not limited to, Fto knocks in fluorescent reporter gene in site, to indicate the reduction of fto gene expression, for screening and establishing treatment human obesity Novel drugs and new method.

9. the feature as described in right 6, it includes but is not limited to fluorescence report that the regulating and controlling sequence of zebra fish fto gene, which can be used for driving, Gene, with prepare transgenosis zebra fish, for screening and establishing the novel drugs and new method for the treatment of human obesity.

10. feature as described in claim 1, the fto gene that the fto gene of zebra fish can be other fish model animals is made To identify gene, for screening and establishing the novel drugs and new method for the treatment of human obesity.