CN108103108A - The preparation and its application of Cebpa gene delection zebra fish mutant - Google Patents
The preparation and its application of Cebpa gene delection zebra fish mutant Download PDFInfo
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Abstract
The invention discloses a kind of preparations and its application of Cebpa gene delections zebra fish mutant；Design is for the TALEN sequences of zebra fish cebpa genes；Its left and right arms plasmid is built, and in-vitro transcription goes out corresponding mRNA；TALEN left and right arms mRNA is mixed and microinjection is into zebra fish fertilized egg；Embryo after injection supports to F0 adult fishes and mates with wild type adult fish, and the embryo for carrying cebpa gene mutations is supported to F1 adult fishes；F1 adult fishes containing cebpa gene mutations mate again with wild type adult fish, and the embryo of generation is supported to F2 adult fishes, and the method that genomic DNA is extracted by cutting fishtail fin is identified, determines the mutant.The present invention by TALEN gene editing technologies, can be rapidly and efficiently establish cebpa gene delection zebra fish strains, can be carried out for treatment leukaemia using the model, liver development is bad and the drug screening work of the diseases such as granulocyte missing.
Description
Technical field
The invention belongs to gene editing technical fields, are related to a kind of Cebpa Gene Deletion zebra fish mediated by TALEN
The preparation and its application of mutant.
Background technology
CCAAT enhancer binding proteins α (CCAAT/enhancer-binding protein α, C/EBP α) is C/EBPs
Important member in transcription factor family, is encoded by single exon, C-terminal have highly conserved DNA binding structural domains and
Dimerization functional domain (basic region and leucine zipper domain, bZIP).The study found that C/EBP α are making
Blood is developed, and is played an important role during liver development and Adipocyte Differentiation etc., and dysfunction is even more and a variety of diseases
The generation and development of disease are closely related.In Patients with Acute Myeloid Leukemia, C/EBP α gene mutation rates are about 5%-14%.
Zebra fish as a kind of emerging model organism, had both " small mode biology " (such as yeast, nematode, drosophila) and
Numerous advantages of " large model biology " (such as mouse)：Zebra fish is easy to laboratory and raises on a large scale into fish length 3-4cm；Once
Mating can produce hundreds of pieces of embryos, facilitate and carry out large-scale chemical mutagen screen mutation and live body high-flux medicaments sifting；From
Development of fertilized ova is short to the sexal maturity individual time, it is only necessary to 2-3 months；Zebra fish is in vitro fertilization, vitro Development of Embryos, early stage
It is transparent, it is easy to real-time monitored and operation；Diplont, the work of zebra fish gene order-checking have been completed, and many signals lead to
Road and important gene are all highly conserved with the mankind, and its nervous system, immune system, cardiovascular system, reproductive system etc.
Development and function also with the mankind there are many common ground, can be as the animal model for studying human developmental and disease.In addition, with
Going deep into for research, many research means and method also all reach its maturity.It can be seen that zebra fish is a kind of ideal grinds
Study carefully the model organism of vertebrate heredity and development.
TALE (Transcription Activator-Like Effector) is by phytopathogen Xanthomonas campestris
(Xanthomonas) one kind of secretion has the albumen of transcriptional activation function, and the albumen is by some very conservative repetition amino
Acid sequence module forms, and each module generally comprises 33-35 amino acid, wherein the 12nd, 13 amino acids species is variable and determines
The specificity that TALE is combined with DNA is determined.One kind is just formd with spy by the way that TALE is merged with restriction enzyme FokI
Strong tools-TALEN (the transcription activator-like effector of specific gene group editting function
nucleases).TALEN is combined with the target site of genome in the cell, and just plays restriction endonuclease after FokI forms dimer
Activity cuts DNA double chain, so as to which DNA double chain be caused to damage.After DNA damage, body can start two kinds of repair mechanisms,
One kind is nonhomologous end engagement mechanisms (Non-homologous End Joining, NHEJ), and another kind is homologous recombination
Repair mechanism (homologous recombination).When the homologous sequence of no external source adds in, cell will start
NHEJ repair mechanisms reconnect the DNA ends of fracture together.But this repair mechanism is a kind of quick, non-essence
True reparation easily occurs wrong (missing/insertion), so as to cause frameshift mutation, thus may finally reach gene knockout
Purpose.As a kind of emerging genome editing technique, TALEN systems have it is easy to operate, the time is short, the advantages such as at low cost.
CRISPR/Cas technologies can complete the DNA identifications of RNA guiding and compile as another genome edit tool
Volume.Although CRISPR/Cas technologies are more flexible in target site design, easy, it is not specifically high, and miss rate is far above
TALEN technologies, and its gene targeting efficiency is also below TALEN technologies.
The content of the invention
It is an object of the invention to overcome above-mentioned the shortcomings of the prior art, a kind of Cebpa mediated by TALEN is provided
The preparation and its application of Gene Deletion zebra fish mutant.Cebpa genes and the generation and development of a variety of diseases are closely related,
The foundation of this model will have good medical research value and commercial application value.
The purpose of the present invention is what is be achieved through the following technical solutions：
The present invention relates to a kind of preparation methods of Cebpa gene delections zebra fish mutant, which is characterized in that the method
Include the following steps：
S1, design are directed to the TALEN sequences of zebra fish cebpa genes；
S2, the left and right arm plasmid for building TALEN, and in-vitro transcription goes out the left and right arm mRNA of TALEN respectively；
S3, the left and right arm mRNA of TALEN are mixed into simultaneously microinjection into zebra fish fertilized egg；
S4, the embryo after injection is supported to F0 adult fishes, and mated with wild type adult fish, the embryo of generation carried out restricted
Inscribe enzyme method and the identification of DNA sequencing method, the embryo for carrying cebpa gene mutations is supported to F1 adult fishes；
S5, to F1 adult fishes cut fishtail fin extracting genomic DNA method identification with the presence or absence of cebpa gene mutations, and
Its cebpa gene mutation type is determined by DNA sequencing method, by the F1 adult fishes containing cebpa gene mutations again with it is wild
Type adult fish mates, and the embryo of generation is supported to F2 adult fishes；
S6, the method identification for extracting genomic DNA by cutting fishtail fin to F2 adult fishes, determine mutation fish system, the mutation fish
System is the Cebpa gene delections zebra fish mutant.
Preferably, in step S1, the left arm sequence of TALEN sequences is as shown in SEQ ID NO.1, the right arm of TALEN sequences
Sequence is as shown in SEQ ID NO.2.
Preferably, treat that embryonic development to 2-3 days, selects the embryo of several pieces of normal developments after injection is further included in step S2
Tire extracts genomic DNA, and PCR amplification is carried out to cebpa genes, and pcr amplification product is reflected by the method for restriction enzyme
Determine the whether effective steps of TALEN.
Preferably, primer sequence such as the SEQ ID NO.3, SEQ ID NO.4 of PCR amplification use are carried out to cebpa genes
It is shown.
Preferably, clone's zebra fish cebpa mutators are further included in step S6 into pCS2+ expression vectors, by glimmering
Light element enzyme reporter assay (luciferase assay) detects whether mutation C/ebp α also have the function of the step of transcriptional activity.
Preferably, further included in step S6 and support the F2 embryos generated for the selfing of cebpa mutant to 4-6 days, utilize Soviet Union
The step of granulocyte developmental state of the method detection cebpa gene knockout embryos of red black dyeing.
The invention further relates to the Cebpa gene delection zebra fish mutant described in a kind of basis preparation method establish
Cebpa gene delections zebra fish model is as treatment leukaemia, liver development is bad and granulocyte deficit disorder
Purposes in medicaments sifting model.
Compared with prior art, the present invention has the advantages that：
1st, the present invention, can be quick by TALEN gene editing technologies, efficiently establishes cebpa gene delection zebra fish
Strain.CRISPR/Cas technology gene targeting efficiencies are slightly below TALEN technologies, and miss rate is higher than TALEN technologies, and this hair
The bright TALEN technologies that are innovatively used in combination can more effectively knock out the cebpa genes encoded by single exon.cebpa
Gene and the generation and development of a variety of diseases are closely related, can carry out the research of relevant disease in a deep going way on the basis of this model,
Such as leukaemia, liver cancer etc., so as to good medical research value.
2nd, the model established using the present invention can be carried out for treatment leukaemia, and liver development is bad and granulocyte lacks
The drug screening work of the diseases such as mistake, so as to possess very promising commercial application value.
Description of the drawings
Fig. 1 is the zebra fish cebpa genes of the present invention and TALEN target site schematic diagrames；Wherein, underscore base is respectively
TALEN left arms and right arm sequence, overstriking base are restriction enzyme SalI restriction enzyme sites；
Fig. 2 is electrophoresis detection result schematic diagram；
Fig. 3 is sequencing result schematic diagram；
Fig. 4 is sudan black coloration result schematic diagram.
Specific embodiment
With reference to embodiment, the present invention is described in detail.Following embodiment will be helpful to those skilled in the art
The present invention is further understood, but the invention is not limited in any way.It should be pointed out that those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it can also make certain adjustments and improvements.These belong to the guarantor of the present invention
Protect scope.
Embodiment
1st, TALEN gene knockouts target site designs
In http：The genome of //asia.ensembl.org/index.html query site zebra fish cebpa genes
DNA sequence dna.According to TALEN design principles, in https：//tale-nt.cac.cornell.edu/ website designs are directed to zebra
The TALEN sequences of fish cebpa genes.The TALEN left and right arms sequences of design are respectively TAL1：T CGAGGGAAATCCAAG(SEQ
ID NO.1)；TAL2：T GTACTCGGTGCTGTT(SEQ ID NO.2).
2nd, TALEN left and right arms mRNA in-vitro transcriptions
Transcribe synthesis TALEN left and right arms mRNA respectively using in-vitro transcription kit.
3rd, the microinjection of zebrafish embryo and target spot validity measure
TALEN left and right arms mRNA is mixed into (final concentration is respectively 150ng/ul), is carried out in fertilized eggs one cell stage micro-
Injection, injection volume 2nl.After injection when embryonic development is small to 48, the embryo of 5 pieces of normal developments is selected, extracts genome
DNA, and PCR amplification is carried out to cebpa genes.Primer is respectively cebpa-Forward：
ATGGAGCAAGCAAACCTCTACGAGG(SEQ ID NO.3)；cebpa-Reverse：
TTAAGCGCAGTTGCCCATGGCTTTG(SEQ ID NO.4).Reaction condition is：94 DEG C of 25min of pre-degeneration；94 DEG C of denaturation
30s, anneal 58 DEG C of 30s, extends 72 DEG C of 1min, expands 35 Xun Huans；72℃10min.10ul PCR products is taken to carry out SalI enzymes
Cut 2 it is small when, the validity of electrophoresis detection TALEN target sites, and to PCR product sequencing analysis.
Fig. 1 is the zebra fish cebpa genes of the present invention and TALEN target sites；Underscore base be respectively TALEN left arms and
Right arm sequence.Red base is restriction enzyme SalI restriction enzyme sites.
4th, the preparation of the person of building F0 at the beginning of cebpa gene knockouts
Microinjection is carried out to zebra fish fertilized egg according to the method for step 3, development respectively cut juvenile fish after 60 days
Tail fin is taken, prepares genomic DNA template, F0 is identified according to the method for step 3, determines the just person of building F0.
5th, F1 mutant screens
It collects the F0 embryos generated that mate with wild-type zebrafish to support to adult fish i.e. F1, clip tail fin carries out genome respectively
Type identifies that screening obtains being capable of the cebpa mutant F1 generations of heredity.
Genomic DNA is extracted to the F1 heterozygotes of foundation, utilizes abrupt climatic change primer cebpa-Forwrd and cebpa-
Reverse carries out PCR amplification, and SalI digestion identifications, such as Fig. 2 are carried out to PCR product, and the results show TALEN systems can have
Effect destroys cebpa genes in genomic level.
Genomic DNA is extracted to the F1 heterozygotes of foundation, utilizes abrupt climatic change primer cebpa-Forwrd and cebpa-
Reverse carries out PCR amplification, and PCR product is sequenced, such as Fig. 3, and the results show TALEN systems cause target gene
Cebpa exons 1s have lacked two bases, and so as to cause the generation of frameshift mutation, the bZIP for destroying C/ebp α PROTEIN Cs end is tied
Structure domain.
6th, F2 mutant screens
It collects the F1 mutant embryo generated that mates with wild-type zebrafish to support to adult fish i.e. F2, clip tail fin carries out respectively
Genome type identifies that screening obtains to stablize the cebpa mutant F2 generations of heredity.
7th, granulocyte developmental state after sudan black dyeing detection cebpa missings
The embryo that the selfing of the cebpa mutant in F2 generations generates was supported to 5 days, and with 4% paraformaldehyde room temperature fix 2 it is small when
Afterwards, granulocyte developmental state in sudan black dyeing detection cebpa deletion homozygotes is carried out.The F2 heterozygotes of foundation are selfed, production
Result such as Fig. 4 that raw embryonic development carried out sudan black dyeing by the 5th day, the result is shown in the homozygote embryos that cebpa is knocked out
Middle granulocyte missing.
Sequence table
<110>Ruijin Hospital, Shanghai Jiao Tong University School of Medicine
<120>The preparation and its application of Cebpa gene delection zebra fish mutant
<130> 2017
<141> 2018-01-30
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
Claims (7)
1. a kind of preparation method of Cebpa gene delections zebra fish mutant, which is characterized in that the described method includes following steps
Suddenly：
S1, design are directed to the TALEN sequences of zebra fish cebpa genes；
S2, the left and right arm plasmid for building TALEN, and in-vitro transcription goes out the left and right arm mRNA of TALEN respectively；
S3, the left and right arm mRNA of TALEN are mixed into simultaneously microinjection into zebra fish fertilized egg；
S4, the embryo after injection is supported to F0 adult fishes, and mated with wild type adult fish, restriction enzyme is carried out to the embryo of generation
Enzyme method and the identification of DNA sequencing method, the embryo for carrying cebpa gene mutations is supported to F1 adult fishes；
S5, to F1 adult fishes cut fishtail fin extracting genomic DNA method identification with the presence or absence of cebpa gene mutations, and pass through
DNA sequencing method determines its cebpa gene mutation type, by the F1 adult fishes containing cebpa gene mutations again with wild type into
Fish mates, and the embryo of generation is supported to F2 adult fishes；
S6, the method identification for extracting genomic DNA by cutting fishtail fin to F2 adult fishes, determine mutation fish system, mutation fish system is
The Cebpa gene delections zebra fish mutant.
2. the preparation method of Cebpa gene delections zebra fish mutant according to claim 1, which is characterized in that step
In S1, the left arm sequence of TALEN sequences is as shown in SEQ ID N0.1, the right arm sequence such as SEQ ID N0.2 institutes of TALEN sequences
Show.
3. the preparation method of Cebpa gene delections zebra fish mutant according to claim 1, which is characterized in that step
Embryonic development is treated to 2-3 days after injection is further included in S2, selects the embryo of several pieces of normal developments, extracts genomic DNA, and it is right
Cebpa genes carry out PCR amplification, and pcr amplification product identifies whether TALEN effectively walks by the method for restriction enzyme
Suddenly.
4. the preparation method of Cebpa gene delections zebra fish mutant according to claim 3, which is characterized in that right
Cebpa genes carry out the primer sequence of PCR amplification use as shown in SEQ ID N0.3, SEQ ID N0.4.
5. the preparation method of Cebpa gene delections zebra fish mutant according to claim 1, which is characterized in that step
Clone's zebra fish cebpa mutators are further included in S6 into pCS2+ expression vectors, it should by luciferase reporting experiment detection
Whether mutation C/ebp α also have the function of the step of transcriptional activity.
6. the preparation method of Cebpa gene delections zebra fish mutant according to claim 1, which is characterized in that step
It is further included in S6 and supports the F2 embryos generated for the selfing of cebpa mutant to 4-6 days, detected using the method for sudan black dyeing
The step of granulocyte developmental state of cebpa gene knockout embryos.
7. the cebpa bases that a kind of preparation method of Cebpa gene delections zebra fish mutant according to claim 1 is established
Because missing zebra fish model is as treatment leukaemia, liver development is bad and the drug screening of granulocyte deficit disorder
Purposes in model.
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| CN114107399A (en) * | 2021-11-09 | 2022-03-01 | 苏州大学 | Construction method and application of tsh beta gene deletion zebra fish model |
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| CN113416752A (en) * | 2021-06-23 | 2021-09-21 | 周娟 | Mog1 gene knockout zebra fish model and application |
| CN114107399A (en) * | 2021-11-09 | 2022-03-01 | 苏州大学 | Construction method and application of tsh beta gene deletion zebra fish model |
| CN114600837A (en) * | 2022-04-15 | 2022-06-10 | 润康生物医药(苏州)有限公司 | Animal model of agranulocytosis, construction method thereof and application of ikzf1 and cmyb in construction of model |
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