CN1590548A - Recombination plasmid of expression bifluorescent gene - Google Patents
Recombination plasmid of expression bifluorescent gene Download PDFInfo
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- CN1590548A CN1590548A CN 03157924 CN03157924A CN1590548A CN 1590548 A CN1590548 A CN 1590548A CN 03157924 CN03157924 CN 03157924 CN 03157924 A CN03157924 A CN 03157924A CN 1590548 A CN1590548 A CN 1590548A
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Abstract
A recombinant plasmid expressing two fluorescent genes contains a universally present promoter, a fluorescent gene able to be linked with and inserted to downstream of said promoter, a skin or muscle specific promoter, and another fluorescent gene able to be linked with and inserted to downstream of said skin or muscle specific promoter. A host cell and a transgenic fish with said plasmid are also disclosed.
Description
Technical field
The present invention relates to a recombinant plasmid, a kind of generation contains the method for genetically engineered fish, host cell and the transgenic animal of this recombinant plasmid.
Background technology
Genetically engineered fish research system utilizes gene to be driven by allos and homologous regulatory element, and produced by composing type or tissue specific expression gene.Controlling element comprises the gene or the like of delta, carp β Actin muscle, salmon tissue protein H3 and the carp alpha globulin of antifreeze protein, mouse metallothionein(MT), chicken.Yet, in genetically engineered fish, use these DNA elements that very big shortcoming is arranged, comprise the low and genetically modified mosaic expression of expression efficiency.
After the medaka ovum is gone in the lac reporter gene microinjection that medaka (mekada) β actin promoter is driven, though express seldom and have a mosaicism of height, but can cause lacZ gene transient expression, even also can express at F1.After people such as Hamada were merged green fluorescent protein and medaka β actin promoter, microinjection also had similar result to the medaka embryo's that fish-egg generated report.(Hamada etc., 1998, MolMarine Biol Biotechnol 7:173-180).
People such as Chi-Yuan Chou have delivered two ends and have connected reverse terminal repeat, make up plasmid with the DNA that increases transgene expression efficient in the medaka body.Transgenosis the 0th generation and after the expression in two generations all be consistence (Chi-Yuan Chou etc., 2001, Transgenic Research 10:303-315).In addition, people such as Chung-Der Hsiao also point out after the reverse terminal repeat of adeno-associated virus (AAV-ITRs) the embedding transgenosis, can make the zebra fish genetic expression unanimity in the 0th generation, and very stable (the Chung-Der Hsiao etc. of the transfer of render transgenic, 2001, DevelopmentalDynamics 220:323-336).
Zebra fish (zebrafish; Danio rerio) is a kind of new model animals of vertebrates developmental biology research.With the zebra fish is that experiment model has several main advantages, for example the obtaining easily of ovum and embryo, embryo's forming process organize clear, ectogenesis, generation time short, and adult fish and juvenile fish all be easy to raise etc.
Existing people utilizes and comprises rat actomyosin light chain enhanser (Moss, J.B. etc., Greenfluorescent protein marks skeletal muscle in murine cell lines and zebrafish.Gene 173,8998,1996), the first type insulin-like growth factor promotor (Chen of zebra fish and Tilapia, J.Y. etc., Isolation and characterization of tilapia (Oreochromismossambicus) insulin-like growth factors gene and proximal promoter region (DNA Cell Biol.17,359-376,1998) etc. various gene promoter imports known fluorogene such as GFP gene (comprising the EGFP gene) in the zebra fish.The purpose of these transgenic experiments all is to can be used for the GFP transgenosis system of analyzing gene expression or the regulating DNA element in the test cdna promotor in order to develop one.
Patent application WO0049150 discloses a kind of by the expressed fluorescence transgenic ornamental fish of single fluorogene (as GFP).But, still untappedly go out any fish that can consistently reach the fluorogene that stably gives expression to two or more.
Summary of the invention
The present invention relates to a recombinant plasmid, comprise: (a) ubiquitous promotor, (b) fluorogene, this gene can be controlled connection and insert under this ubiquitous promotor and swim, (c) skin specificity or muscle specific promotor, and (d) another fluorogene, this gene can be controlled connection and insert under this skin specificity or the muscle specific promotor and swim, wherein, this ubiquitous promotor and skin specificity or muscle specific promotor have reverse property (adverse directional property), and ubiquitous promotor and skin specificity or muscle specific promotor lay respectively at the upstream of this fluorogene and this another fluorogene, can be for the directivity of described genetic transcription to have.
The present invention also relates to a kind of host cell that contains plasmid of the present invention.
In addition, the invention still further relates to a method that produces genetically engineered fish, this method comprises:
A) plasmid with the present invention imports in fish egg cell or the embryonic cell, and
B) make ovum or embryonic cell grow adult fish, wherein the present invention's plasmid imports in the genome of this fish.
The invention further relates to a method that can produce the genetically engineered fish of expressing two kinds of fluorogenes simultaneously, this method comprises the following step:
A) in suitable restriction enzyme cleavage site, plasmid with restriction enzyme cutting the present invention, to obtain two plasmid fragment I and II, wherein this plasmid I contain in the present invention's the plasmid defined a) and b) fragment, and this plasmid II contains defined c in the present invention's the plasmid) and d) fragment;
B) respectively the plasmid A of this step a) and B are imported in fish egg cell or the embryonic cell; And
C) make this fish give expression to plasmid I and II simultaneously.
The present invention also relates to a kind of transgenic animal that transformed via the plasmid of being invented.
Description of drawings
Fig. 1 shows the therefore photo of genetically engineered fish of two kinds of fluorescent bases of expression.
Fig. 2 shows that plasmid A is connected the schema that the back produces plasmid C with plasmid B.
Embodiment
The invention provides and a kind ofly the gene of two or more (for example green and red fluorescence) can be imported the interior recombinant plasmid of fish body.These fishes can present uniformity and intensive fluorescence under general light source.
The invention provides a recombinant plasmid, comprise: (a) ubiquitous promotor, (b) fluorogene, this gene can be controlled connection and insert under this ubiquitous promotor and swim, (c) promotor of skin specificity or muscle specific, and (d) another fluorogene, this gene can be controlled connection and insert under this skin specificity or the muscle specific promotor and swim, wherein the promotor of this ubiquitous promotor and skin specificity or muscle specific has reverse property, and the promotor of ubiquitous promotor and skin specificity or muscle specific lays respectively at the upstream of this fluorogene and this another fluorogene, can be for the directivity of described genetic transcription to have.
Mention term " upstream " in the literary composition and reach " downstream ", meaning is promptly when reference direction is defined as direction from the initiator codon of fluorogene to terminator codon, certain a bit is positioned at and the unidirectional end of reference direction, just " downstream " end opposite with reference direction that is referred to as this point then is " upstream " of this point.
According to the present invention, the plasmid expression that its ubiquitous startup subsystem is used to drive the present invention goes out fluorogene.The promotor of ubiquitous promotor and skin specificity or muscle specific has reverse property, and ubiquitous promotor is positioned at the upstream of this fluorogene, to have the directivity that can transcribe for fluorogene.Ubiquitous promotor preferably is selected from the group of being made up of beta-actin, elongation-1-α, 18S-rDNA or 5S-rDNA.
According to the present invention, the startup subsystem of skin specificity or muscle specific is used for driving fluorescent base and therefore expresses.The promotor of skin specificity or muscle specific is positioned at the upstream of this fluorogene, to have the directivity that can supply fluorogene to transcribe.The promotor of tool skin specificity or muscle specific preferably is selected from by the heavy chain of α-Ji Dongdanbai, troponin (troponin) T, TnC, actomyosin, Cytokeratin II C type (cytokeratin type II C) or group that S-100 formed.
According to the present invention, any fluorogene all can be inserted into the upstream of the promotor of plasmid of the present invention.Fluorogene preferably is selected from the group that green, redness, yellow or blue-fluorescence gene are formed.These fluorogenes can buy on the market, for example, and can be available from Clonteh LaboratoriesInc., Lightools Research and BD Biosciences Pharmingen etc.With the group that is selected from by green and red fluorescence genomic constitution serves as preferred.
People such as Hiroshi Otsuki point out that when using ubiquitous promotor, rice crop comprises that green fluorescence (Hiroshi Otsuki all can appear in all parts of callus (calli), January 12-16,2002, Plant, Animal ﹠amp; Microbe Genomes X Conference).According to the present invention, the green fluorescence gene can be controlled and connect and insert under the present invention's the ubiquitous promotor and swim.The initial system of green fluorescent protein (GFP) separates from certain jellyfish (Aequorea victoria), and can buy on the market.GFP is different with other Bioluminescent reporter molecule, as long as it touches UV-light or blue light, just can give out bright green glow.The green glow that is distributed is because the energy of aequorin (aequorin) is transferred to due to the GFP.GFP is that 238 amino acid constitute, molecular weight is the protein of 28kDa, and it is that 395nm, less important peak are the single emission peak of 470nm and 509nm that its spectrum mainly absorbs the peak.The advantage of GFP is that its fluorescence do not have a species dependency, and need not lean on substrate, cofactor or other protein can send green glow.Existing people successfully utilizes several to give expression to GFP as host living beings such as intestinal bacteria, yeast, mammalian cell, insect cell and vegetable cell and cell.
According to the present invention, the red fluorescence gene can be available from BD Bioscience Clontech.When enforcement was of the present invention, pDsRed2-1 system was used as the source of red fluorescence gene.The pDsRed2-1 codified goes out DsRed2, and it is a kind of DsRed mutation through design, comparatively fast maturation and less non-specific aggregation.DsRed2 stems from the red fluorescent protein (drFP583 of mushroom coral; Matz, M.V. waits (1999) Nature Biotech.17:969-973.), it contains a series of reticent base pair and changes, select to prefer to the mammalian cell camber with the mankind's codon and express be consistent (Haas, J. wait (1996) Curr.Biol.6:315-324.).In mammalian cell is cultivated, when the DsRed2 constitutive expression, in 24 hours of transfection, just can detect the cell that gives out redness by fluorescent microscope.In bacterium of expressing DsRed1 and mammalian cell system, observe big soluble protein aggregation, in the cell of expressing DsRed2, can reduce many dramatically through regular meeting.Very fast maturation and more diffluent red fluorescent protein also can make host cell than the tool tolerance.The sign of not having tangible survival rate minimizing with DsRed2 mammalian cells transfected culture, and in the cell strain of those tests, the form that cell occurred (as sticking power, light refraction) of expression DsRed2 and growth characteristics are the same with the control group that untransfected is crossed.PDsRed2-1 is a DsRed2 carrier that does not contain promotor, can be used for monitoring and inserts the different promoters in the multiple cloning site (MCS) and the Transcription of promotor/enhanser combination.The sequence that will be positioned at the DsRed2 upstream is changed into the total translation initiation position (Kozak, M. (1987) Nucleic Acids Res.15:8125-8148.) of Kozak, to increase the translation efficiency in the eukaryotic cell.3 ' end at the bootable DsRed2 mRNA of SV40 polyadenylic acid signal in DsRed2 gene downstream is done suitable processing.Carrier mainly contains one can be used to the Puc replication orgin and a f1 starting point of making single-stranded dna of breeding for expressing SV40 starting point that the antigenic mammalian cell of SV40T duplicates, one in intestinal bacteria.A kind of have a neomycin resistance box (Neo
r) can utilize G418 to select the mammalian cell of stable transfection, this box contains Xin Meisu/Kanr gene of SV40 early promoter, Tn5 and from the polyadenylic acid signal of simplexvirus thymidine kinase (HSV TK) gene.In intestinal bacteria, the bacterium promotor of box upstream can give expression to Kanr.
According to the present invention, the standard molecule technology of recombinant plasmid this area, to contain therefore plasmid of ubiquitous promotor and fluorescent base, and contain the promotor of skin specificity or muscle specific and another fluorescent base therefore plasmid combine and constituted.When enforcement is of the present invention, cut the plasmid that contains ubiquitous promotor and green fluorescence gene with restriction enzyme, to obtain the fragment of a 4.7kb.The plasmid that contains the promotor of skin specificity or muscle specific and red fluorescence gene also obtains the fragment of one section 7.5kb with the restriction enzyme cutting.Via engagement step 4.7kb fragment and the 7.5kb fragment that is produced linked together.
The invention provides a kind of host cell that contains plasmid of the present invention.According to the present invention; can utilize some host systems to hold the present invention's plasmid; these host systems comprise the microorganisms such as bacterium, plasmid or cosmid DNA expression vector that transform with recombinant phage; yeast with the Yeast expression carrier conversion; the insect cell system that infected with virus expression carrier; the vegetable cell system that infected with virus expression carrier or bacterial expression vector, or the zooblast system etc., but be not limited only to this.
The invention provides a kind of transgenic animal that transformed with plasmid of the present invention.According to the present invention, these transgenic animal can be any animals of conveniently obtaining, the Mammals of non-human for example, as the rodent and the fish that are used for the lab investigation process are exactly an example.For example use via the tradition of microinjection or injection recombinant vectors gene manipulation techniques easily again, plasmid of the present invention is imported in the animal body, just can obtain the present invention's transgenic animal easily.Gene can directly or indirectly enter via the precursor of transfered cell in the cell or all cells of animal.Gene manipulation techniques comprises that classical cross-breeding, in vitro fertilization, importing may be integrated in the karyomit(e) or become the recombinant DNA molecules of the DNA of extrachromosomal replication.Transgenic animal are preferred with the genetically engineered fish, and with genetically engineered fish selected in medaka, zebra fish, seven color angle fish, goldfish, clanging or clanking sound fish, kind porgy, peacock fish, imperial fish, bright and beautiful carp, bucket fish or other aquarium fish for more preferably.According to the present invention, the genetically engineered fish of being invented can give expression to a kind of green and red color of mixing.
According to one embodiment of the invention, plasmid of the present invention can be used for producing the fish of expressing a kind of fluorescence, doping fluorescent and expressing different fluorescence simultaneously.Just express a kind of fluorogene, the plasmid that utilizes suitable restriction enzyme cutting to be invented is to produce the plasmid fragment that contains a kind of fluorogene.The one section plasmid that is generated is imported in the ovum or embryonic cell of fish, to obtain expressing a kind of fish of fluorogene.Just express doping fluorescent, the plasmid of being invented is imported in fish-egg or the embryonic cell, to obtain expressing the fish of doping fluorescent.Just express different fluorescence simultaneously, the different plasmids that will contain different fluorogenes import in fish-egg or the embryonic cell, to be expressed the fish of different fluorogenes simultaneously.
Preferably, plasmid of the present invention can be used to produce and expresses single green fluorescence gene, single red fluorescence gene, doping green and red fluorescence gene or express green simultaneously and the fish of red fluorescence gene.Just express the fish of green fluorescence,, contain ubiquitous promotor and control the plasmid of green fluorescence gene that connects and insert the downstream of this ubiquity promotor to obtain one with the plasmid of restriction enzyme in the cutting the present invention of suitable position.The plasmid that is produced is imported in fish-egg or the embryo, just can give expression to green fluorescence.Just express the fish of red fluorescence, with the plasmid of restriction enzyme in suitable position cutting the present invention, contain the promotor of skin specificity or muscle specific and control the plasmid of red fluorescence gene that connects and insert the promotor downstream of this skin specificity or muscle specific to obtain one, and, just can give expression to red fluorescence with in the plasmid importing fish egg cell or embryonic cell that are produced.
According to a preferred embodiment of the invention, the invention provides a method that can produce the genetically engineered fish of expressing doping fluorescent, this method comprises the plasmid that a) will invent and imports in fish egg cell or the embryonic cell, and b) allow ovum or embryonic cell grow adult fish, wherein the plasmid of being invented is imported in the genome of fish, to obtain giving expression to the fish of doping fluorescent.Fluorogene serves as preferred with the group that is selected from green, redness, yellow or blue-fluorescence gene and is formed, and is selected from group that green or red fluorescence gene formed then more preferably as fluorogene.
According to a preferred embodiment of the invention, the invention provides a method that can produce the genetically engineered fish of expressing two kinds of different fluorescence simultaneously, this method comprises the following step:
A) with restriction enzyme behind the plasmid of suitable restriction enzyme cleavage site cutting claim 1, can obtain two plasmid fragments of I and II, wherein plasmid I contain fragment that claim 1 defines a) and b), plasmid II contains the fragment c that claim 1 defines) and d);
B) step a) described each plasmid A and plasmid B are imported respectively in fish egg cell or the embryonic cell,
C) make aforesaid fish give expression to plasmid I and II simultaneously.
According to the present invention, the fish from the method manufacturing of invention is come out can give expression to two kinds of different fluorescence simultaneously.With the plasmid of restriction enzyme in suitable position cutting the present invention, to obtain two plasmids, one of them plasmid contains ubiquitous promotor and one to be controlled and connects and insert the fluorogene in the downstream of this ubiquity promotor, and another plasmid then contains the promotor of skin specificity or muscle specific and another to be controlled and connect and insert the fluorogene in this skin specificity or muscle specific promotor downstream.Then, two plasmids that produced are imported respectively in the fish, to give expression to different fluorogenes separately.Fluorogene is selected from group that green, redness, yellow or blue-fluorescence gene form for preferred, and it is then more preferred to be selected from the group of being formed that contains green and red fluorescence gene as fluorogene.
According to a preferred embodiment of the invention, can demonstrate redness and green fluorescence simultaneously from the resulting fish of the inventive method.Preferably, can utilize technology known in the art to come further breeding and propagation, to obtain the fish of unanimity and stably express redness and green fluorescence from the resulting fish of the inventive method.
The following example further specifies the present invention, but non-desire restriction the present invention's scope.
Embodiment
Embodiment 1 construction comprises the plasmid of GFP sequence
Separate tool skin specificity or muscle specific and energy ubiquity ground and express the cloned strain of zebra fish cDNA.The separation of cDNA clone and sequencing such as Gong, Z. etc. are at Gene201, described in the 87-98 (1997).Basically, the random cdna cloned strain is the cDNA gene pool that is selected from zebrafish embryo and adult fish, and comes partly each cDNA cloned strain of sequencing via single sequencing reaction.This section partial sequence is used for confirming the potential function and the tissue specificity of sequencing cloned strain subsequently.
As shown in Figure 2, plasmid A is the structure view of a structure plasmid.GFP is driven by the promotor of ubiquity.After plasmid was by NotI and the linearizing of PstI restriction enzyme, (dNTP) filled and led up its sticky end with deoxyribonucleotide, reclaimed the fragment of 4.7kb again in agarose gel electrophoresis.This plasmid illustrates the composition that makes up plasmid according to the present invention.
One section plasmid that comprises the red fluorescent protein sequence of embodiment 2 construction
As embodiment 1, plasmid B is for making up the structure view of plasmid as shown in Figure 2.DsRed is driven by the promotor of skin specificity or muscle specific.After plasmid was by the linearizing of PstI restriction enzyme, (dNTP) filled and led up its sticky end with deoxyribonucleotide, reclaimed the fragment of 7.5kb again in agarose gel electrophoresis.This plasmid illustrates the composition that makes up plasmid according to the present invention.
Embodiment 3 makes up plasmid by connecting plasmid A and B
As shown in Figure 2, plasmid C one makes up the structure of plasmid, is expressed as the linearizing fragment of being cut via restriction enzyme NotI, or via restriction enzyme NotI or linearizing fragment that SalI cut.This plasmid is used for the composition of the structure plasmid of transgenosis according to the present invention's explanation.
Embodiment 4 introduces plasmid and fluorescence microscopy
Under artificial condition illumination in 14 hours and ten hours dark and raise zebra fish with Tetramin (TetraGermany).Be fertilized to collect ovum in 30 minutes and remove and link filament.Zygote is remained on 6 ℃ up to till the dna fragmentation microinjection, and this dna fragmentation (as with the linearization plasmid C of NotI cutting or with the linearization plasmid C of NotI and SalI cutting) is before first division, with 10 μ g/ml concentration injection tenuigenin.In distilled water, cultivate through the injection ovum at 26 ℃.(MZAPO, Leica Germany) observe the embryo with dissecting stereoscopic microscope under bright territory.The luminous system in dark territory of detecting green and/or red fluorescence carries out with the stereoscopic microscope that is equipped with GFP and/or red fluorescent protein.Camera with the controller that has ISO 400 egative films and egative film time exposure is taken pictures.
Embodiment 5: manufacturing can be expressed the transgenic zebrafish of two kinds of fluorescence
The zebra fish breeding
Male and the female zebra fish in one pond is supported fishbowl at one 60 * 20 * 30 centimetres, temperature is located at the irradiation cycle of 28.5 ℃ and 14 hours.Feed fish twice with fairy shrimp every day, afterwards, picks out severally to strong male and female zebra fish, and places an incubation slot of 30 * 10 * 20 centimetres with the net of collection ovum.In order to set up the transgenosis strain, with a pair of genetically engineered fish and wild breeding in 22 * 14 * 13 centimetres cylinder.
The screening of transgenosis and original seed genetically engineered fish (transgenic founder)
Collect zygote with plastic capillary, and be placed in the shelf.In the glass needle of opening 10 μ m, fill linearizing plasmid solution, and fix with mineral oil.Then with the dna sample microinjection of volume 2-4nl to unicellular zygote.Zygote being placed on of injecting contained in the culture dish of Methylene blue solution of lower concentration and hatched, and place and set 28 ℃ incubator.In the time of the 3rd day, use fluorescent microscope promptly to can be observed the embryo who gives expression to fluorescence.After five days, the fluorescence zebra fish is moved in the fish jar of raising.Just attainability maturation after 12 weeks.
The generation that the kind system of fluorescence transgenic zebrafish transmits
To have luciferase expression infer the original seed genetically engineered fish and wild strain is hybridized.Transgenosis F2 (s-generation) is by hybridization mutually between two fluorescence F1, and this fish all can be observed the expression of fluorescence throughout one's life.
Embodiment 6: the potential application of fluorescence genetically engineered fish
The fluorescence genetically engineered fish can be used as aquarium fish on market.Can carry out the breeding of GFP transgenic animal and wild fish or another kind of genetically engineered fish and develop stable transgenosis strain.Via isolating more zebra fish gene promoter, for example have eyes specificity, bone specificity, tail specificity etc., with and/or raise these transgenic zebrafishes via traditional method, just can produce a greater variety of fluorescence transgenic zebrafishes.
Because blue fluorescent protein (BFP) gene, yellow fluorescence protein (YFP) gene and dark green fluorescin (CFP) gene all can buy in Clonetech, therefore utilize identical technology just can produce the fluorescence fish of multiple color.For example, have GFP under the specific promotor of eyes, have the genetically engineered fish that has YFP under the promotor that has BFP under the specific promotor of skin and have muscle specific having, following multiple fluorescence color will occur: green eye, blue skin and yellow eyes.To have after specific promotor of different tissues and fluorescence protein gene reconfigure, can create the genetically engineered fish that more different colours change.Utilize homologue to express two or more different fluorescin, just can create a kind of middle shade.
By using heavy metal (for example cadmium, cobalt, chromium) or hormone (for example oestrogenic hormon, androgen or other steroid hormone) inducible promoter, can develop and a kind ofly be used for that monitoring environment pollutes and assessment is human drinks and water is ploughed the biological sensing system of the water quality of usefulness.In a such biological sensing system, when the pollutent environment in water as heavy metal and oestrogenic hormon (or derivatives thereof) and so on reaches thresholding concentration, genetically engineered fish just can send green fluorescence (or other color, decide on the fluorescence protein gene that is used).This biological sensing system is better than traditional analytical procedure, and is fast because of its speed, can be visible and can directly differentiate specific compound in the complex mixture that environment was found in the water outlet, and is easy to carry or more do not need the accessory instrument.In addition, biological sensing system also can provide the direct information of relevant bio-toxicity, and it is biological can decompose and reproducible.
The environmental monitoring of number of substances, also can a kind ofly have codified and go out genetically engineered fish, just can be observed fish shown particular color that goes out in environment then by createing by the gene of the fluorescin of the different colours that promotor drove that various materials are responded.Perhaps, also can transform some fishes by distinctive carrier, these fishes can be mixed then and be used in the shoal of fish of monitoring of environmental, and observe the expressed color of coming out of each fish.
In addition, the fluorescence genetically engineered fish should also have value in the market of scientific research material, because they can be used for the research as the embryo aspect of following the trail of cell strain and cell migration.The cell of the genetically engineered fish of expression GFP also can be used as cell and the genetic marker in Transplanted cells and the nuclear transplantation experiment.
The present invention successfully prove can be on zebra fish two fluorogenes of construction, this also can be used for the fish of other kind, for example medaka, goldfish, the carp that comprises bright and beautiful carp, loach fish, Tilapia, kiss prelarva, catfish, scalare, seven color angle fish, eel, bucket fish, goby, vest fish, peacock fish, Xiphophorus helleri (red sword), hatchet fish, Morley fish, fresh water shark etc.
The above person is only for the partly explanation of preferred embodiment of the present invention, so the variation of the equivalent method of all application specification sheets of the present invention and claims ought to be included in the present invention's the claim.
Claims (12)
1. recombinant plasmid, comprise: (a) ubiquitous promotor, (b) fluorogene, this gene can be controlled the downstream that connects and insert this ubiquitous promotor, (c) promotor of skin specificity or muscle specific, and (d) another fluorogene, this gene can be controlled the downstream that connects and insert this skin specificity or muscle specific promotor, wherein this ubiquitous promotor and skin specificity or muscle specific promotor have reverse property, and this ubiquitous promotor and skin specificity or muscle specific promotor lay respectively at the upstream of this fluorogene and this another fluorogene, can be for the directivity of described genetic transcription to have.
2. the recombinant plasmid of claim 1, the wherein group formed of the optional free beta-actin of this ubiquitous promotor, elongation-1-α, 18S-rDNA or 5S-rDNA.
3. the recombinant plasmid of claim 1, the wherein optional free α-Ji Dongdanbai of the promotor of this skin specificity or muscle specific, troponin (troponin) T, TnC, myosin heavy chain, Cytokeratin II C type (cytokeratin type II C) or group that S-100 formed.
4. host cell, it comprises the plasmid of claim 1.
5. method that produces genetically engineered fish, this method comprises:
A) plasmid with claim 1 imports in fish egg cell or the embryonic cell, and
B) make this ovum or embryonic cell grow adult fish, wherein the plasmid of claim 1 imports in the genome of this fish.
6. the genetically engineered fish of claim 5, the wherein group formed of the optional free medaka of this fish, zebra fish, seven color angle fish, goldfish, Medaka fish, kind porgy, peacock fish, imperial fish, carp or bucket fish.
7. genetically engineered fish, it comprises (a) ubiquitous promotor, (b) fluorogene, this gene can be controlled the downstream that connects and insert this ubiquitous promotor, (c) skin specificity or muscle specific promotor, and (d) another fluorogene, this gene can be controlled the downstream that connects and insert this skin specificity or muscle specific promotor, wherein this ubiquitous promotor and skin specificity or muscle specific promotor have reverse property, and this ubiquitous promotor and skin specificity or muscle specific promotor lay respectively at the upstream of this fluorogene and this another fluorogene, can be for the directivity of described genetic transcription to have.
8. the genetically engineered fish of claim 7, wherein this fluorogene is selected from the group of being made up of green, red, yellow or blue-fluorescence gene.
9. the genetically engineered fish of claim 8, wherein this fluorogene is selected from the group of being made up of green and red fluorescence gene.
10. the method for the genetically engineered fish of two kinds of different fluorogenes is expressed in a manufacturing simultaneously, and this method comprises the following step:
A) at suitable restriction enzyme cleavage site, plasmid with restriction enzyme cutting claim 1, to obtain two plasmid fragment I and II, wherein this plasmid I contain claim 1 defined a) and b) fragment, plasmid II then contains the defined c of claim 1) and d) fragment;
B) respectively the plasmid I of this step a) and II are imported in fish egg cell or the embryonic cell, and
C) make this fish give expression to plasmid I and II simultaneously.
11. the method for claim 10, the wherein group formed of optional free green, red, yellow of this fluorogene or blue-fluorescence gene.
12. the method for claim 11, the wherein group formed of optional free green of this fluorogene or red fluorescence gene.
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| CNB031579248A CN1297664C (en) | 2003-08-29 | 2003-08-29 | Recombination plasmid of expression bifluorescent gene |
| HK05107856A HK1075914A1 (en) | 2003-08-29 | 2005-09-07 | Recombinant plasmids expressing two flurorescent genes |
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| CNB031579248A CN1297664C (en) | 2003-08-29 | 2003-08-29 | Recombination plasmid of expression bifluorescent gene |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006122443A1 (en) * | 2005-05-16 | 2006-11-23 | Zhongshan University | The method for knocking-out the fish gene |
| CN102936596A (en) * | 2012-08-23 | 2013-02-20 | 北京大学 | Medaka ovary structural protein gene promoter and applications thereof |
| CN103540611A (en) * | 2013-09-26 | 2014-01-29 | 马明 | Method for producing color-changeable light-induced fluorescent spectacular fish |
| CN106399370A (en) * | 2016-09-12 | 2017-02-15 | 中国海洋大学 | Method for establishing stable transgenic flounder embryo cell strain |
| CN107828822A (en) * | 2017-11-06 | 2018-03-23 | 南京中医药大学 | The production method of the fluorescence fish of epidermis changeable colour based on eucaryotic cell structure tension variation |
| US20200113159A1 (en) * | 2017-03-30 | 2020-04-16 | GloFish, LLC | Transgenic rainbow shark |
| US20210051927A1 (en) * | 2018-01-10 | 2021-02-25 | GloFish, LLC | Transgenic rainbow shark |
| US11716975B2 (en) | 2014-11-07 | 2023-08-08 | Glofish Llc | Blue transgenic fluorescent ornamental fish |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7135613B1 (en) * | 1999-02-18 | 2006-11-14 | The National University Of Singapore | Chimeric gene constructs for generation of fluorescent transgenic ornamental fish |
| WO2002088368A1 (en) * | 2001-05-02 | 2002-11-07 | Institute Of Molecular Agrobiology | Spatial and temporal control of gene expression in the zebrafish using baculovirus |
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2003
- 2003-08-29 CN CNB031579248A patent/CN1297664C/en not_active Expired - Fee Related
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2005
- 2005-09-07 HK HK05107856A patent/HK1075914A1/en not_active IP Right Cessation
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106399370A (en) * | 2016-09-12 | 2017-02-15 | 中国海洋大学 | Method for establishing stable transgenic flounder embryo cell strain |
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| US20200113159A1 (en) * | 2017-03-30 | 2020-04-16 | GloFish, LLC | Transgenic rainbow shark |
| CN107828822A (en) * | 2017-11-06 | 2018-03-23 | 南京中医药大学 | The production method of the fluorescence fish of epidermis changeable colour based on eucaryotic cell structure tension variation |
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| US20210051927A1 (en) * | 2018-01-10 | 2021-02-25 | GloFish, LLC | Transgenic rainbow shark |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1297664C (en) | 2007-01-31 |
| HK1075914A1 (en) | 2005-12-30 |
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