CN1304571C - Method for breeding transgenic zebra fish and its special plasmid - Google Patents
Method for breeding transgenic zebra fish and its special plasmid Download PDFInfo
- Publication number
- CN1304571C CN1304571C CNB2003101004529A CN200310100452A CN1304571C CN 1304571 C CN1304571 C CN 1304571C CN B2003101004529 A CNB2003101004529 A CN B2003101004529A CN 200310100452 A CN200310100452 A CN 200310100452A CN 1304571 C CN1304571 C CN 1304571C
- Authority
- CN
- China
- Prior art keywords
- apoaequorin
- zebra fish
- expression vector
- recombinant expression
- generation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Abstract
The present invention discloses a method for breeding a transgenic zebra fish and a special plasmid thereof. The method for breeding an apoaequorin transgenic zebra fish of the present invention comprises the following steps: (1) an apoaequorin recombinant expression vector is constructed, and the apoaequorin recombinant expression vector comprises an apoaequorin coding gene, a GATA2 regulation and control sequence of a zebra fish, and a GFP expression frame guided by an EF-1 alpha gene regulation sequence of xenopus laevis; (2) the recombinant expression vector in step (1) is used to convert the zygotes of the zebra fish to obtain an F0 generation; (3) the F0 generation mates with a wild zebra fish to obtain the transgenic zebra fish which can express apoaequorin. The apoaequorin transgenic zebra fish of the present invention performs important functions on biological development research, the living body calcium image research of zebra fishes, etc. and has important theoretical and actual significance.
Description
Technical field
The present invention relates to a kind of method and special-purpose plasmid thereof of cultivating genetically engineered fish, particularly a kind of method and special-purpose plasmid thereof of cultivating transgenic zebrafish relates in particular to a kind of method and special-purpose plasmid thereof of the apoaequorin of cultivation transgenic zebrafish.
Background technology
Calcium ion is counted as the second messenger in the cell, at cell, transmits signal of interest between histoorgan, and the communication between mediation internal body and outside atmosphere is coordinated normal development, the functionating of biont by the expression of the quick transcription factor gene of regulation and control calcium.
Apoaequorin (E14215.1 GI:5708898 in Genbank) is a kind of luminescent protein of finding in the jellyfish, it and a kind of cofactor coelenterazine are combined into aequorin, aequorin produces coelenteramide with can decomposing automatically after calcium ion combines, apoaequorin, carbonic acid gas and a kind of blue-fluorescence.Can this optical signal be amplified and note by micro-imaging equipment.Therefore aequorin can be used as the calcium ion acceptor and detects calcium ion concn and variation.Mature methods is that apoaequorin and coelenterazine are injected directly in the viable cell or zygote to be studied now, detects the calcium ion signal by the photon picture reproducer.But along with cellular segregation, the apoaequorin in the daughter cell and coelenterazine can be fewer and feweri, detectable calcium signal can be more and more a little less than.
Transgenic technology can change a foreign gene in the host genome over to, and the foreign gene that changes over to can be expressed as host's endogenous gene in host cell.Zebra fish is the desirable vertebrates material that is used for transgenosis.In general, the transgenic technology of comparative maturity has the microinjection of linearizing recombinant plasmid and has the infecting of retrovirus of foreign gene.These two kinds of methods all are competent in the transgenic zebrafish kind system that obtains some amount, and wherein the former is relatively simple.
Expression of gene mainly is subjected to the cis-acting elements of the upstream of gene or intron (as promotor, enhanser etc.) regulation and control, the expression of foreign gene in transgenic animal is so equally, and its expression is subjected to being implemented in the control of the promotor in the expression vector.
The innovation and creation content
The method that the purpose of this invention is to provide the transgenic zebrafish that a kind of cultivation can stably express apoaequorin.
A kind of method of cultivating the apoaequorin transgenic zebrafish may further comprise the steps:
(1) make up the apoaequorin recombinant expression vector, described apoaequorin recombinant expression vector comprises GATA2 regulating and controlling sequence of apoaequorin encoding gene, zebra fish and the GFP expression cassette that is guided by Africa xenopus EF-1 α gene regulating sequence;
(2) transform zebra fish zygote with the recombinant expression vector in the step (1), obtain F0 generation;
(3), obtain to express the transgenic zebrafish of apoaequorin with the zebra fish mating of F0 generation with wild-type.
Wherein, the transgenic zebrafish that obtains in the step (3) can obtain the homozygote of transgenic zebrafish by traditional breeding method.The apoaequorin recombinant expression vector can pass through the apoaequorin encoding gene, GATA2 regulating and controlling sequence (the Anming M. of zebra fish, Hong T., Bruce A.O., Micheal J.F., Shuo L.. (1997) .Promoter analysis in living zebrafish embryos identifies a cis-acting motifrequired for neuronal expression of GATA-2.Proc.Natl.Acad.Sci..94,6267-6272.), Africa xenopus EF-1 α gene (XEX) regulating and controlling sequence (Johnson A.D., Krieg P.A.. (1995) .A Xenopus laevis gene encoding EF-1alpha S, the somatic form ofelongation factor 1 alpha:sequence, structure, and identification ofregulatory elements required for embryonic transcription.Dev Genet.17 (3), 280-90.) and GFP expression cassette (Amsterdam A., Lin S., Hopkins N.. (1995) .The Aequoreavictoria green fluorescent protein can be used as a reporter in live zebrafishembryos.Dev Biol.171 (1) 123-9.) inserts carrier pGEM-7Zf (-) (X65311.2GI:6687355in the Genbank) and obtains.The apoaequorin recombinant expression vector can import zebra fish zygote by several different methods, as microinjection, protoplastis fusion method, high voltage electric perforation method etc., is preferably microinjection.
The present invention is directed to along with apoaequorin in the cellular segregation daughter cell and coelenterazine meeting problem less and less, cultivate the apoaequorin transgenic zebrafish by the method for gene recombination, can stably express apoaequorin.Comprise the GATA2 promotor in the apoaequorin recombinant expression vector of the present invention, the transgenic zebrafish of preparing is expressed apoaequorin at muscle and neural system respectively.For the ease of identifying the apoaequorin transgenic zebrafish, all comprise GFP expression cassette on each expression vector by the guiding of Africa xenopus EF-1 α gene (XEX) regulating and controlling sequence, in the transgenic zebrafish of success, can be observed the green fluorescence that general is expressed.
Apoaequorin transgenic zebrafish of the present invention will be studied at biological development, zebra fish live body calcium picture plays important effect in studying, and have important significance for theories and practical significance.Apoaequorin transgenic zebrafish of the present invention can be at specific histoorgan and the specific synthetic in a large number apoaequorin of developmental stage, can be used on active somatic cell, organ even individual integral level research, to illustrate the effect of calcium signal in processes such as growth, growth to calcium signal (calcium picture).Apoaequorin transgenic zebrafish of the present invention and existing mutant zebra fish can obtain the relation of relevant specific gene sudden change and calcium abnormal signal by genetic breeding, on the mechanism of genetic diseases is set forth and treated important meaning are arranged.
Description of drawings
Fig. 1 is the physical map that can express the recombinant expression vector of apoaequorin
Fig. 2 is the in situ hybridization result who contains the apoaequorin transgenic zebrafish of GATA2 promotor
Fig. 3 is the expression photo that contains the apoaequorin transgenic zebrafish GFP of GATA2 promotor
Embodiment
The apoaequorin transgenic zebrafish is expressed in embodiment 1, cultivation
Cultivate the method for expressing the apoaequorin transgenic zebrafish, may further comprise the steps:
(1) makes up the recombinant vectors that to express apoaequorin
PolyA (pA) and apoaequorin coding region fragment that the clone is obtained are inserted among the carrier pGEM-7Zf (-), obtain plasmid pGEM-Aeq-pA.With XhoI and BamHI digested plasmid pGATA2-GM2, obtain the GATA2 promoter fragment of about 2.1kb; With XhoI and SacI digested plasmid pBKS-GM2, obtain the XEX-GM2-pA fragment of about 1.6kb; With BamHI and SacI digested plasmid pGEM-Aeq-pA, obtain the fragment of about 3.9kb; Connect this three fragments, obtain recombinant vectors pGATA2-Aeq-GM2.
The physical map of constructed recombinant expression vector of expressing apoaequorin as shown in Figure 1, the GATA2 regulating and controlling sequence that comprises zebra fish, also comprise a GFP expression cassette, be used to screen transgenic zebrafish by the guiding of Africa xenopus EF-1 α gene (XEX) regulating and controlling sequence.
(2) recombinant expression vector pGATA2-Aeq-GM2 transforms zebra fish zygote
The linearizing recombinant plasmid of microinjection is fed to sexual maturity in zebra fish zygote, obtains F0 generation.
(3) acquisition of transgenic zebrafish
With the zebra fish mating of F0 generation with wild-type, whether have GFP to express to identify GFP male F1 generation by observing its filial generation embryo F1 generation, the result obtains the positive F1 generation that general is expressed GFP as shown in Figure 3.(with apoaequorin is template to use the apoaequorin antisense RNA probes of digoxigenin labeled again, the RNA sequence that obtains by 3 '-＞5 ' direction in-vitro transcription) does whole embryo in situ hybridization according to a conventional method, check the apoaequorin expression of F1 for the embryo, the result as shown in Figure 2, finally identify the transgenic zebrafish of expressing apoaequorin, among the figure, the black-dyeing position is neural neurone.Obtain the homozygote of transgenic zebrafish again by traditional breeding method.
Claims (5)
1, a kind of method of cultivating the apoaequorin transgenic zebrafish may further comprise the steps:
(1) make up the apoaequorin recombinant expression vector, described apoaequorin recombinant expression vector comprises GATA2 regulating and controlling sequence of apoaequorin encoding gene, zebra fish and the GFP expression cassette that is guided by Africa xenopus EF-1 α gene regulating sequence;
(2) transform zebra fish zygote with the recombinant expression vector in the step (1), obtain F0 generation;
(3), obtain to express the transgenic zebrafish of apoaequorin with the zebra fish mating of F0 generation with wild-type.
2, method according to claim 1, it is characterized in that: in the described step (1), GATA2 regulating and controlling sequence and the Africa xenopus EF-1 α gene regulating sequence of apoaequorin encoding gene, zebra fish are inserted among the carrier pGEM-7Zf (-), obtain described apoaequorin recombinant expression vector.
3, method according to claim 1 and 2 is characterized in that: described apoaequorin recombinant expression vector imports zebra fish zygote by microinjection, protoplastis fusion method, high voltage electric perforation method, obtains F0 generation.
4, method according to claim 3 is characterized in that: described apoaequorin recombinant expression vector imports zebra fish zygote by microinjection, obtains F0 generation.
5, a kind of plasmid of cultivating the apoaequorin transgenic zebrafish, it comprises GATA2 regulating and controlling sequence of apoaequorin encoding gene, zebra fish and the GFP expression cassette that is guided by Africa xenopus EF-1 α gene regulating sequence.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101004529A CN1304571C (en) | 2003-10-16 | 2003-10-16 | Method for breeding transgenic zebra fish and its special plasmid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101004529A CN1304571C (en) | 2003-10-16 | 2003-10-16 | Method for breeding transgenic zebra fish and its special plasmid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1607252A CN1607252A (en) | 2005-04-20 |
| CN1304571C true CN1304571C (en) | 2007-03-14 |
Family
ID=34755973
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2003101004529A Expired - Fee Related CN1304571C (en) | 2003-10-16 | 2003-10-16 | Method for breeding transgenic zebra fish and its special plasmid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1304571C (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102363792A (en) * | 2011-11-11 | 2012-02-29 | 中国水产科学研究院淡水渔业研究中心 | Lentivirus method for preparing IGF2b transgenic fish |
| CN105925607A (en) * | 2016-06-02 | 2016-09-07 | 贵州医科大学 | Establishment method for transgenic zebrafish model with glucose-6-phosphate dehydrogenase deficiency |
| CN106035233B (en) * | 2016-06-22 | 2019-10-29 | 贵州医科大学 | Transgenic zebrafish model and construction method with g6pd1303-1497 site deletion |
| CN106070063B (en) * | 2016-07-07 | 2019-03-05 | 贵州医科大学 | Transgenic zebrafish system and its method for building up with abcb4 |
| CN112430624B (en) * | 2020-11-25 | 2023-08-18 | 汕头大学 | Zebra fish muscle specific induction type expression vector and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001092300A2 (en) * | 2000-06-01 | 2001-12-06 | Institut Pasteur | Chimeric gfp-aequorin as bioluminescent ca++ reporters at the single cell level |
-
2003
- 2003-10-16 CN CNB2003101004529A patent/CN1304571C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001092300A2 (en) * | 2000-06-01 | 2001-12-06 | Institut Pasteur | Chimeric gfp-aequorin as bioluminescent ca++ reporters at the single cell level |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1607252A (en) | 2005-04-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6380458B1 (en) | Cell-lineage specific expression in transgenic zebrafish | |
| EP1571222A2 (en) | Fluorescent genetic fragments and the correspondant transgenic fish | |
| US20150166615A1 (en) | Method and uses for Bombyx mori silk fibroin heavy chain mutation sequence and mutant | |
| CN104673815B (en) | Compound piggyBac recombinant vectors and its preparation method and application | |
| Long et al. | FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori | |
| CN1304571C (en) | Method for breeding transgenic zebra fish and its special plasmid | |
| CN1263850C (en) | Method for breeding transgenic zebra fish and its special plasmid | |
| Stern | Transgenic tools for targeted chromosome rearrangements allow construction of balancer chromosomes in non-melanogaster Drosophila species | |
| Hyodo-Taguchi et al. | Phenotypic rescue of the albino mutation in the medakafish (Oryzias latipes) by a mouse tyrosinase transgene | |
| CN110117617A (en) | A kind of method preparing the lethal silkworm strain of female embryo and in-between strain and construct | |
| CN103232977A (en) | Application of phiC31 recombinase system and piggyBac transposon and fixed point transgenetic system of silkworm and preparation method of fixed point transgenetic system | |
| CN1297664C (en) | Recombination plasmid of expression bifluorescent gene | |
| Meredith | 13 Genetic methods for studying ion channel function in physiology and disease | |
| CN102559691B (en) | Silkworm sex chromosome linked gene PdpI and recessive lethal mutation gene thereof and application of silkworm sex chromosome linked gene PdpI and recessive lethal mutation gene in silkworm sex ratio control | |
| CN110904114B (en) | Application of Gnb2 gene coding sequence and Gnb2 gene coding sequence in constructing mouse model | |
| JP4431739B2 (en) | Use of DNA encoding the silkworm kynurenine oxidase gene | |
| Kobayashi et al. | Rescue of the Aojuku white-egg translucent (w-3ol) Bombyx mori mutant by transgenic expression of the wild-type Bmwh3 gene | |
| JP5794620B2 (en) | Arylalkylamine-N-acetyltransferase gene and use thereof | |
| CN114606270B (en) | Construction method of Mia3 conditional gene knockout mouse model | |
| CA2450452C (en) | Transgenic animal transformed using green fluorescent protein gene | |
| CN113564205B (en) | Construction method of balanced chromosome animal model | |
| JP2004321173A (en) | Recombinant plasmid expressing two fluorescent gene | |
| CN116941573A (en) | Construction method and application of spontaneous mutation Hr gene introduced inbred line mouse model | |
| Meng et al. | Tissue-specific expression of GFP reporter gene in germline driven by GATA-2 promoter and enhancers in zebrafish | |
| CN103882013A (en) | Insect gender modulating construct, modulating method, and application of the construct |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070314 Termination date: 20091116 |