CN101962660B - Tgf2 transposon-based fish transgenic method and vector thereof and preparation method for vector - Google Patents
Tgf2 transposon-based fish transgenic method and vector thereof and preparation method for vector Download PDFInfo
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Abstract
The invention discloses a Tgf2 transposon-based fish transgenic method. The method can realize efficient transgene by injecting transgenic donor plasmids and two vectors of Tgf2 transposase mRNA to fertilized eggs of 1 to 2 cell stages of fish together. The transgenic donor plasmids contain left and right arms of goldfish Tgf2 transposon, fish promoter DNA sequences and target genes. The Tgf2 transposase mRNA is obtained by transferring in vitro of auxiliary plasmids containing goldfish Tgf2 transposase reading frame DNA sequences. Compared with the conventional fish transgenic technology, the method has the advantages of high integration efficiency, high load capacity, generality, safety and high efficiency.
Description
Technical field
The present invention relates to set up a kind of fish transgenosis method, also disclose the transgenic donor plasmid that this method relates to and the construction process of transposase mRNA in-vitro transcription helper plasmid simultaneously based on the Tgf2 transposon, and the application in zebra fish and cultured fishes.The invention belongs to fish genetically engineered field.
Background technology
China is aquaculture big country, and for a long time, China's cultured output accounts for 2/3rds of Gross World Product, and China strides forward towards aquaculture power fast at present.China's aquaculture is of a great variety; The breed kind that comprises fish, shrimp, shellfish, algae reaches kind more than 200; At present; The water industry breeding exploitation of China is main with traditional breeding technologies such as seed selection, hybridization, chromosome engineerings mainly, and new varieties speed is slow, and China's aquatic products breeding fraction of coverage is merely about 20% at present.Be that physilogical characteristics such as main aquatic products kind reproductivity is strong and in vitro fertilization, external hatching all very help transgeneic procedure with fish; Make fish become most suitable transgenic animal experiment material and genetic improvement object; Transgenic technology is that the rapid breeding of the numerous aquatic products objects of China brings opportunity, is the important outlet that improves China's aquatic products breeding fraction of coverage.China's the eighties in last century is promptly developed the transgenic carp and the transgenic Oncorhynchi of quick growth, and the theoretical method of genetically engineered fish development all has world lead level with the achievement that obtains.
Transgenic is with the method in the dna fragmentation quiding gene group of carrying gene.Through observing the change of transgenic organism objective trait, the contriver can obtain genetically modified mutant, for breeding of new variety.Fish, the most frequently used transgenic method is that the linearizing DNA that carries foreign gene is passed through in the means transfered cells such as liposome, electroporation or microinjection DNA not to be gone in the genome through studying machine-processed random integration fully aware of so far yet.This method has a lot of shortcomings: 1) integration efficiency is low, is difficult to effectively in the less shrimps of the less fish of pelagic egg, adhesive egg fish, ovulation amount and ovum, to carry out transgenic breeding; 2) abnormal rate is high, and this method need be injected more a large amount of plasmids, and the existence of a large amount of exogenous plasmid copies can make that development of fertilized ova is unusual, causes a large amount of deformities; 3) in most cases linearizing DNA can form the series connection repetition of multiple copied, i.e. concatenate (concatamer), and it is difficult to normal physiological situation of simulation native gene; 4) concatenate is easy to reorganization takes place between different transgenic copies when going down to posterity and causes disappearance (deletion), or modifies the genetically modified stably express of influence owing to methylate; 5) owing to utilize the very difficult position of confirming in the genome of transgenic insertion place of this method, therefore also can't make assessment near the karyomit(e) environment the insertion site.Compare with simple linearizing DNA, retroviral vector can be integrated into genome by render transgenic more efficiently, but its main deficiency is: 1) loading capacity is little, promptly can carry the limited length of exogenous dna fragment; 2) host also can be to the virus modification that methylates, render transgenic expression silencing; 3) prepare viral complex operation; 4) though to be used for genetically modified be replication-defective virus mostly, security is still the vigilant problem of needs.Therefore, in fish, need high, the loading capacity transgenic novel method big and safe and convenient to use of the new integration efficiency of exploitation.
Transposon is one type of ability changes on position in host genome a Mobile Genetic Elements, and the process of its change on position is called as swivel base.According to the difference of swivel base mode, transposon can be divided into DNA transposon and retrotransposon two big classes.The swivel base of DNA transposon is followed " cutting off-paste " mechanism, and promptly under the catalysis of transposase, the DNA transposon cuts off and be inserted into new genome position from the original position.Transposon can not only import the acceptor gene group with foreign gene as the transgenic instrument, and simultaneously, it is again because can make the native gene inactivation become " favorite " in the forward genetics research through insertion mutagenesis at random.
The gene transfer system of DNA transposon is a transgenic technology of new generation, but has the DNA transposon of autonomous transposition activity rarely found in vertebrates.1996; The Japan scholar vertebrates hAT family transposon that discovery has natural radioactivity in the blue or green Medaka of albefaction (Oryziaslatipes) first; Be blue or green Medaka Tol2 transposon, this transposon has carried out widespread use in model organism zebra fish transgenic, gene and promotor aspect catching.2008, the contriver found that a new Tgf2 transposon with natural radioactivity extensively exists in some goldfish kinds of China, and goldfish Tgf2 transposon is the second routine vertebrates transposon of finding so far.Identical with blue or green Medaka Tol2 transposon element; Goldfish Tgf2 transposon contains that the latter end inversion repeats, inferior terminal repetition, intercalary inversion repeats and active transposase gene, goldfish Tgf2 and blue or green Medaka Tol2 transposon the latter end inversion repeat and transposase gene there is some difference on encoding.Fish high-efficient transgenic method involved in the present invention is based upon on the goldfish Tgf2 transposon basis exactly.
Summary of the invention
The present invention is exactly in order to address the above problem; Overcoming the most frequently used transgenic method is through in the means transfered cells such as liposome, electroporation or microinjection with the linearizing DNA that carries foreign gene; Integration efficiency is low; Be difficult to effectively in the less shrimps of the less fish of pelagic egg, adhesive egg fish, ovulation amount and ovum, to carry out transgenic breeding, abnormal rate is high, and in most cases linearizing DNA can form the series connection repetition of multiple copied; Concatenate is in the instability of expression of gene, and difficult definite transgenic is inserted the position in the genome of place.And the retroviral vector loading capacity is little, and the host also can be to the virus modification that methylates, and the render transgenic expression silencing prepares viral complex operation, the problem of security hidden danger.Therefore, the present invention is intended to make up a kind of fish high-efficient transgenic novel method based on transposon.Deriving from the transgenic donor plasmid of goldfish Tgf2 transposon left and right arms, and be aided with Tgf2 transposase mRNA, co-injected is gone into fish 1-2 cell stage zygote, can realize high-efficient transgenic.In fish, need high, the loading capacity transgenic novel method big and safe and convenient to use of the new integration efficiency of exploitation.
The technical problem that will solve required for the present invention can realize through following technical scheme.
As first aspect of the present invention; A kind of fish transgenosis method based on the Tgf2 transposon is characterized in that, is through carrier goal gene to be injected into fish; Said carrier comprises: transgenic donor plasmid and fish Tgf2 transposase 5 ' add cap mRNA; With the transgenic donor plasmid, be aided with fish Tgf2 transposase 5 ' and add cap mRNA co-injected and go into fish 1-2 cell stage zygote, promptly realize high-efficient transgenic.
As second aspect of the present invention, a kind of carrier of the fish transgenosis method based on the Tgf2 transposon is characterized in that,
Said carrier comprises: transgenic donor plasmid and fish Tgf2 transposase 5 ' add cap mRNA;
Said transgenic donor plasmid comprises: fish Tgf2 transposon left and right arms, fish promoter DNA sequence and goal gene;
Further, it is Tgf2 transposase mRNA in-vitro transcription helper plasmid that said fish Tgf2 transposase 5 ' adds cap mRNA, is obtained by the helper plasmid in-vitro transcription again.
Further, said fish Tgf2 transposon left and right arms is from goldfish Tgf2 transposon.
Further, said fish promoter DNA sequence is zebra fish β-actin promotor.
Further, said goal gene is jellyfish green fluorescent protein (eGFP) gene.
Further, the structure of said in-vitro transcription helper plasmid is that Tgf2 transposase mRNA reading frame dna sequence dna is connected the helper plasmid of construction expression transposase with the SP6 promotor.
As the third aspect of the invention, a kind of preparation method of carrier of the fish transgenosis method based on the Tgf2 transposon is characterized in that,
(1) structure of transgenic donor plasmid: the utilization round pcr is cloned zebra fish β-actin promoter sequence respectively; Make up the expression casette of jellyfish green fluorescent protein (eGFP); Said destination gene expression box is cloned between the left and right arms sequence of Tgf2 transposon through gene clone technology, makes up the fish transgenosis donor plasmid;
(2) Tgf2 transposase 5 ' adds cap mRNA:Tgf2 transposase mRNA reading frame dna sequence dna and is connected with the Sp6 promotor; Structure can be expressed the helper plasmid of transposase, adopts Sp6 mMessagemMachine kit (Ambion) test kit to carry out the in-vitro transcription that Tgf2 transposase 5 ' adds cap mRNA again.
In order to realize that with the goal gene stable integration transgenic donor plasmid of the present invention comprises the left and right arms of Tgf2 transposon in the fish genome, comprise inverted repeats and inferior terminal repeat, can discern by transposase, promote the swivel base of dna fragmentation between left and right arms.The present invention utilizes the DNA recombinant technology, the destination gene expression box is cloned between the left and right arms sequence of Tgf2 transposon, has made up the donor plasmid of fish transgenosis.
Transposon generation swivel base also needs Tgf2 transposase 5 ' to add the participation of cap mRNA except that the left and right arms sequence that needs the Tgf2 transposon.Cis element in the transposase identification left and right arms; And realization left and right arms sequence and inner dna fragmentation generation swivel base thereof; For the stable gene of realizing swivel base is incorporated in the fish genome, the present invention is cloned into the Tgf2 transposase gene in another carrier, and trip adds the SP6 promotor above that; Thereby make up the helper plasmid that can carry out Tgf2 transposase vivoexpression, in-vitro transcription acquisition Tgf2 transposase 5 ' adds cap mRNA again.
This based on the Tgf2 transposon; Through the transgenic donor plasmid, be aided with fish Tgf2 transposase 5 ' and add cap mRNA, co-injected is gone into fish 1-2 cell stage zygote; Not only can in fish, realize high-efficient transgenic, and guarantee the genetic stability that transgenic is integrated.
Beneficial effect of the present invention:
The present invention guarantees the high effective integration of external source goal gene in the fish genome by Tgf2 transposon transgenic donor plasmid and the two fish high-efficient transgenic method that constitutes jointly of Tgf2 transposase mRNA.The present invention compared with prior art has safe, efficient and general characteristics, and its efficient is nearly 10 times of traditional plasmid injecting method, for fish gene functional research and transgenic breeding provide new way.
Description of drawings
Further specify the present invention below in conjunction with accompanying drawing and embodiment.
Fig. 1 is the pT2-EF1a-eGFP plasmid map.
Fig. 2 is the pTgf2-EF1a-eGFP plasmid map.
Fig. 3 is pTgf2-zf β-actin-eGFP transgenic donor plasmid collection of illustrative plates.
Fig. 4 is the in-vitro transcription helper plasmid pCS2-CMV-gfTP collection of illustrative plates of Tgf2 transposase mRNA.
Fig. 5 is 36 hours the visible transmission photo of zebrafish embryo fertilization of injection pTgf2-zf β-actin-eGFP+Tgf2 transposase mRNA.
Fig. 6 is 36 hours the visible transmission photo of zebrafish embryo fertilization of injection pTgf2-zf β-actin-eGFP+Tgf2 transposase mRNA.
Fig. 7 be behind the pond crucian carp embryo fertilization of injection pTgf2-zf β-actin-eGFP+Tgf2 transposase mRNA 6 days the visible transmission photo.
Fig. 8 is 6 days a visible transmission photo behind the pond crucian carp embryo fertilization of injection pTgf2-zf β-actin-eGFP+Tgf2 transposase mRNA.
Embodiment
In order to make technique means of the present invention, creation characteristic, to reach purpose and effect and be easy to understand and understand,, further set forth the present invention below in conjunction with concrete diagram.
Below in conjunction with the practical implementation case, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, unspecified technology is a routine techniques.
The clone of embodiment 1, fish Tgf2 transposon left and right arms
Experiment is taken from Feng Qiao culture of ornamental fish field, Nanhui, Shanghai with coloured glaze gold goldfish (Carassius auratus var.) strain.Fresh or the 95% alcohol preservation tail fin in the clip 0.1g left and right sides adds 400 μ L STE damping fluids (30mM Tris-HCl, pH8.0,200mM EDTA, 50mM NaCl).Add final concentration behind the mixing and be respectively 2% SDS and the Proteinase K of 400 μ g/ml, place 55 ℃ of effects of constant temperature shaking table 4h.Add the saturated NaCl of 400ul and the mixing that overturns; Add the abundant mixing of 400ul chloroform again, 4 ℃, 12, the centrifugal 20min of 000rpm; Draw supernatant and add isopyknic isopropanol precipitating DNA; Use 70% washing with alcohol of-20 ℃ of precoolings again, drying at room temperature 5min, coloured glaze gold goldfish genomic dna is dissolved in the aseptic double-distilled water subsequent use.
According to acquired goldfish Tgf2 transposon sequence (GenBank accession number: HM146132), design primer SEQ ID No:1, SEQ ID No:2, SEQ ID No:3 and SEQ ID No:4 respectively.With coloured glaze gold goldfish genomic dna is template, utilizes SEQ ID No:1 (band SpeI restriction enzyme site) and SEQ ID No:2 (band XhoI restriction enzyme site) combination of primers, carries out the pcr amplification of Tgf2 transposon left arm dna sequence dna; Utilize SEQ ID No:3 (band BglII restriction enzyme site) and SEQ ID No:4 primer (band KpnI restriction enzyme site) combination, carry out the pcr amplification of Tgf2 transposon right arm dna sequence dna.The PCR response procedures is: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ prolong 5min.Amplified production is a damping fluid with TBE, after electrophoretic separation under the voltage of 5V/cm through 1.2% sepharose; The purpose fragment is reclaimed in rubber tapping, will reclaim product and be connected in the pMD-19T carrier (TaKaRa), and change bacillus coli DH 5 alpha over to; The picking positive colony, being inoculated into penbritin content is in the liquid LB substratum of 50 μ g/ml, 37 ℃ of shaken overnight are cultivated; Get 200 μ l bacterium liquid glycerine and preserve, and be sent to Shanghai living worker's biotechnology ltd and check order.Sequence results shows; Said Tgf2 transposon left arm dna sequence dna; Be made up of 220 Nucleotide, (the GenBank accession number: the Nucleotide of left arm HM146132) (referring to SEQ ID No:5) has at least 95% homology for this Tgf2-L220 sequence and goldfish Tgf2 transposon sequence; Said Tgf2 transposon right arm dna sequence dna is made up of 185 Nucleotide, and (the GenBank accession number: the Nucleotide of right arm HM146132) (referring to SEQID No:6) has at least 95% homology for this Tgf2-R185 sequence and goldfish Tgf2 transposon sequence.
The structure of embodiment 2, transgenic donor plasmid pTgf2-zf β-action-eGFP
Get the above-mentioned glycerine that comprises the left and right arm sequence plasmid of goldfish Tgf2 transposon respectively and preserve DH5 α bacterium liquid 2 μ L; Being inoculated into 5ml penbritin content respectively is in the liquid LB substratum of 50 μ g/ml; 37 ℃ of shaken overnight are cultivated, and take out test kit (day root) for a short time with plasmid and extract plasmid.The plasmid that comprises goldfish Tgf2 transposon left arm sequence carries out enzyme with restriction enzyme SpeI and XhoI and cuts; The double digestion product is through 1.2% sepharose; With TBE is damping fluid, and after electrophoretic separation under the voltage of 5V/cm, rubber tapping is reclaimed the purpose fragment and is dissolved in the distilled water; Subsequent use in-20 ℃ of preservations, obtain Tgf2-L220 double digestion DNA product.The plasmid that comprises goldfish Tgf2 transposon right arm sequence is cut by restriction enzyme BglII and KpnI enzyme, and the purpose fragment is reclaimed in rubber tapping after electrophoretic separation, obtains Tgf2-R185 double digestion DNA product.
PT2-EF1a-eGFP plasmid (Fig. 1) is carried out SpeI and XhoI double digestion, and enzyme is cut product through 1.2% sepharose, is damping fluid with TBE, and after electrophoretic separation under the voltage of 5V/cm, rubber tapping is reclaimed the purpose fragment and is dissolved in the distilled water.Tgf2-L220 double digestion DNA product and pT2-EF1a-eGFP plasmid double digestion product are connected into pTgf2AL220 with the T4DNA ligase enzyme, and change bacillus coli DH 5 alpha over to, the picking positive colony is sent to Shanghai living worker's biotechnology ltd and carries out sequence verification.Be inoculated into the correct positive colony of sequence in the liquid LB substratum that penbritin content is 50 μ g/ml; 37 ℃ of shaken overnight are cultivated; For a short time take out test kit (day root) with plasmid and extract plasmid, carry out BglII and KpnI double digestion, then Tgf2-R185 double digestion product is connected with the T4DNA ligase enzyme with pTgf2AL220 plasmid double digestion product; And change bacillus coli DH 5 alpha over to; Extract the positive colony plasmid,, obtain comprising the pTgf2-EF1a-eGFP plasmid (Fig. 2) of goldfish Tgf2 left and right arms through sequence verification.
Clip zebra fish (Danio rerio, Shanghai Ocean University's aquatic products and the C202 of life institute) tail fin extracts genomic dna, and is subsequent use in-20 ℃ of preservations.Design primer SEQ ID No:7 (band BamHI restriction enzyme site) and SEQ ID No:8 (band XhoI restriction enzyme site) carry out pcr amplification at the zebra fish genome, and the amplified reaction program is: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 150s, 35 circulations; 72 ℃ prolong 5min.Amplified production is a damping fluid with TBE, after electrophoretic separation under the voltage of 5V/cm through 1.2% sepharose; The purpose fragment is reclaimed in rubber tapping, will reclaim product and be connected to respectively in the pMD-19T carrier (TaKaRa), and change bacillus coli DH 5 alpha over to; The picking positive colony, being inoculated into penbritin content is in the liquid LB substratum of 50 μ g/ml, 37 ℃ of shaken overnight are cultivated; Get 200 μ l bacterium liquid glycerine and preserve, and be sent to Shanghai living worker's biotechnology ltd and check order.The positive colony plasmid that sequence is correct carries out BamHI and XhoI double digestion, and enzyme is cut product through 1.2% sepharose; With TBE is damping fluid; After electrophoretic separation under the voltage of 5V/cm, the purpose fragment is reclaimed in rubber tapping, obtains zebra fish zf β-action promotor double digestion DNA product.
The pTgf2-EF1a-eGFP plasmid is carried out BamHI, XhoI double digestion; Enzyme is cut product through 1.2% sepharose, is damping fluid with TBE, after electrophoretic separation under the voltage of 5V/cm; The purpose fragment is reclaimed in rubber tapping; Obtain pTgf2-eGFP plasmid double digestion product, and then be connected with the T4 ligase enzyme, and change bacillus coli DH 5 alpha over to zebra fish β-action promotor double digestion DNA product; The picking positive colony carries out sequence verification and obtains pTgf2-zf β-actin-eGFP transgenic donor plasmid (Fig. 3).
The in-vitro transcription of embodiment 3, Tgf2 transposase mRNA (5 ' adds cap)
According to acquired information; Tgf2 transposase expression amount in sophisticated ovary is the highest; Get coloured glaze gold goldfish mature ovarian tissue; Utilize TRIZOL (Invitrogen) reagent to extract total RNA, be reversed to 3 ' end strand cDNA with the reversed transcriptive enzyme among TaKaRa RNAPCR Kit (AMV) Ver.3.0, subsequent use in-20 ℃ of preservations.Design SEQ ID No:9 (band BamHI restriction enzyme site) and the positive anti-primer of SEQ ID No:10 (band XbaI enzyme cutting site), amplification Tgf2 transposase (gfTP) coding region total length (1734bp), amplified production is through 1.2% sepharose; With TBE is damping fluid, and after electrophoretic separation under the voltage of 5V/cm, the purpose fragment is reclaimed in rubber tapping; To reclaim product and be connected in the pMD-19T carrier (TaKaRa), and change bacillus coli DH 5 alpha over to, the picking positive colony; Being inoculated into penbritin content is in the liquid LB substratum of 50 μ g/ml; 37 ℃ of shaken overnight are cultivated, and get 200 μ l bacterium liquid glycerine and preserve, and be sent to Shanghai living worker's biotechnology ltd and check order.Sequencing result is got amplified production and is directly carried out BamHI, XbaI double digestion with after expection is consistent, and enzyme is cut product through 1.2% sepharose, is damping fluid with TBE, and after electrophoretic separation under the voltage of 5V/cm, the purpose fragment is reclaimed in rubber tapping, and is subsequent use in-20 ℃ of preservations.
To pCS2+ plasmid BamHI, XbaI double digestion, enzyme is cut product through 1.2% sepharose, is damping fluid with TBE, and after electrophoretic separation under the voltage of 5V/cm, the purpose fragment is reclaimed in rubber tapping, is dissolved in the distilled water.BamHI, the XbaI double digestion product of gfTP are connected with the T4 ligase enzyme with pCS2+ double digestion plasmid, and change bacillus coli DH 5 alpha over to, the picking positive colony is got 200 μ L bacterium liquid glycerine and is preserved, and carries out sequence verification.The picking positive colony is inoculated in the liquid LB substratum that penbritin content is 50 μ g/ml; 37 ℃ of shaken overnight are cultivated; For a short time take out test kit (day root) with plasmid and extract plasmid, obtain the in-vitro transcription helper plasmid pCS2-CMV-gfTP (Fig. 4) of Tgf2 transposase mRNA.Helper plasmid pCS2-CMV-gfTP adopts Sp6 mMessage mMachine kit (Ambion) test kit to carry out Tgf2 transposase 5 ' and adds the synthetic of cap mRNA after the NotI linearizing.
Embodiment 4, the transgenic effect of Tgf2 transposase mRNA mediation donor plasmid in zebra fish
The raising of zebra fish: experiment is raised in the circulation aquarium with zebra fish, and male and female are separately temporarily supported, and feed twice every day, once be the halogen worm, hello artificial diet once, and homo(io)thermism is in 28 ℃, illumination 14h, dark 10h.Lay eggs and last evening raun and milter are matched in same little aquarium, the centre separates with transparent baffle.Morning on the same day was taken out baffle plate by its natural spawning in experiment.
Microinjection: the mixing solutions that respectively adds cap mRNA with 0.3 * Danieau buffer (contain 0.1% phenol red) preparation final concentration for the Tgf2 transposase 5 ' of the pTgf2-zf β of 50ng/ μ l-actin-eGFP transgenic donor plasmid (Fig. 3) and helper plasmid pCS2-CMV-gfTP (Fig. 4) in-vitro transcription.What microinjection was used is the microinjection instrument of the PV830 Pneumatic Pico Pump model of U.S. WPI company, and a whole set of microinjection system drives through nitrogen pressure.Microinjection is used pin to draw the pin device to draw as the 1mm glass capillary through microinjection and is formed, and it is most advanced to remove glass needle with tweezers before using.Mixing solutions with transgenic plasmid solution and Tgf2 transposase mRNA during injection adds in the microinjection pin; And be expelled in zebra fish 1～2 cell stage embryo's the yolk (near the cell position); Each embryo's injection 1nl solution, the ID of transgenic donor plasmid and Tgf2 transposase mRNA respectively is about 50pg.After injection finishes, zygote is positioned over normal development, regularly (every) observation luciferase expression situation under fluorescent microscope in 28 ℃ the embryo medium at a distance from 6 hours.
As a result one: referring to Fig. 5 and Fig. 6; 103 of embryo's survivals in 36 hours of injection pTgf2-zf β-actin-eGFP+Tgf2 transposase mRNA have 89 of green fluorescences (Fig. 5), and the fluorescence rate is 86.4%; Have the zebra fish prelarva of fluorescence to have 38 after 6 days, integration rate is 42.6%.As a result two: 147 of another injection groups embryo survivals in 36 hours, 79 of green fluorescences are arranged, the fluorescence rate is 53.7%; Have the zebra fish prelarva of fluorescence to have 27 after 6 days, integration rate is 34.1%.
Embodiment 5, the transgenic effect of Tgf2 transposase mRNA mediation donor plasmid in pond crucian carp
Test used parent population and take from Shanghai Ocean University Qingpu fish breeding testing station.Select well-developed 2 age pond crucian carp (Carassius auratus gibelio) raun 30 tails and 2 ages male wild carp 18 tails; Play the pin of hastening parturition for pond crucian carp ♀ 3 tails; First pin: luteotropin releasing hormone d-ala analog (LRH-A) 4 μ g/kg, second pin: L2 analogue 6 μ g/kg+HCG1000U/kg; Pond crucian carp ♂ 1 tail is only injected the 2nd pin, and dosage reduces by half.Carried out artificial insemination in second day, zygote sticks on the plastic culture dish, and each petridish bottom is with mark stroke " rice " word line, so that lattice, and numbering.The sticking ovum of each petridish is too not close, avoids adhesion between ovum and ovum as far as possible.Several minutes zygote can be carried out microinjection after water-swelling.Finish writing injection plasmid, time and dosage in injection petridish side.Microinjection instrument system 2 covers are injected by 2 skilled operator, and 2 people are responsible for insemination according to ejection situation, only squeeze a small amount of ovum insemination at every turn, and the residue ovum remains in the parent subsequent use, and parent population places the inflation bucket.2 people are responsible for changing water (per 4 hours) and choose dead ovum (after 24 hours, change and choose clean dead ovum before the water), until membrane.After injection finishes, zygote is placed normal development under the room temperature, regularly (every at a distance from 6 hours) observes the luciferase expression situation under fluorescent microscope.
As a result one: referring to Fig. 7 and Fig. 8, injection pTgf2-zf β-actin-eGFP+Tgf2 transposase mRNA, each embryo's injection 1nl Tgf2 mRNA solution, the ID of transgenic donor plasmid and Tgf2 transposase mRNA respectively is about 50pg.74 of injection pond crucian carp embryo survivals in 24 hours have 51 of green fluorescences, and the fluorescence rate is to have the pond crucian carp prelarva of fluorescence that 38 (Fig. 7) are arranged after 68.9%, 6 day, and integration rate is 51.4%.As a result two: 126 of another injection groups pond crucian carp embryo survivals in 24 hours, 76 of green fluorescences are arranged, the fluorescence rate is 60.3%; Have the pond crucian carp prelarva of fluorescence to have 69 after 6 days, integration rate is 54.7%.Basically about 5%, the efficient of the said transgenic method of contriver is nearly 10 times of traditional plasmid injecting method to the integration rate of conventional linear plasmid injection.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the specification sheets just explains principle of the present invention; The present invention also has various changes and modifications under the prerequisite that does not break away from spirit and scope of the invention, and these variations and improvement all fall in the scope of the invention that requires protection.The present invention requires protection domain to be defined by appending claims and equivalent thereof.
Claims (4)
1. fish transgenosis method based on the Tgf2 transposon; It is characterized in that; Be through carrier goal gene to be injected into fish, said carrier comprises: transgenic donor plasmid and fish Tgf2 transposase 5 ' add cap mRNA, with the transgenic donor plasmid; Be aided with fish Tgf2 transposase 5 ' and add cap mRNA co-injected and go into fish 1-2 cell stage zygote, promptly realize high-efficient transgenic;
Said transgenic donor plasmid comprises: fish Tgf2 transposon left and right arms, fish promoter DNA sequence and goal gene;
Said fish Tgf2 transposon left and right arms all increases from the genomic Tgf2 transposon of goldfish, and the left arm nucleotides sequence of said Tgf2 transposon is classified as shown in the SEQ ID NO:5, and the right arm nucleotides sequence of said Tgf2 transposon is classified as shown in the SEQ ID NO:6;
It is through making up the in-vitro transcription helper plasmid of Tgf2 transposase mRNA, being obtained by the helper plasmid in-vitro transcription that said fish Tgf2 transposase 5 ' adds cap mRNA;
This helper plasmid comprises a Tgf2 transposase reading frame full length DNA; Said Tgf2 transposase reading frame full length DNA is through extracting total RNA of goldfish mature ovarian tissue; Be reversed to 3 ' end strand cDNA again, and adopt primer shown in SEQ ID No:9 and the SEQ ID No:10 to carry out the pcr amplification acquisition;
Said goldfish is a coloured glaze gold goldfish (Carassius auratus var.).
2. the fish transgenosis method based on the Tgf2 transposon according to claim 1 is characterized in that, said fish promoter DNA sequence is zebra fish β-actin promotor.
3. the fish transgenosis method based on the Tgf2 transposon according to claim 1 is characterized in that said goal gene is the jellyfish green fluorescence protein gene.
4. the fish transgenosis method based on the Tgf2 transposon according to claim 1; It is characterized in that; The structure of said in-vitro transcription helper plasmid is that Tgf2 transposase mRNA reading frame dna sequence dna is connected with the SP6 promotor, makes up the helper plasmid that in-vitro transcription Tgf2 transposase 5 ' adds cap mRNA.
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| CN103540611B (en) * | 2013-09-26 | 2015-09-30 | 马明 | A kind of method of producing changeable colour light induced fluorescence aquarium fish |
| CN103710342A (en) * | 2013-12-18 | 2014-04-09 | 浙江农林大学 | Artificially induced MLE (Mariner-Like Element) transposon kit and application thereof |
| CN103981204B (en) * | 2014-03-24 | 2016-05-04 | 上海海洋大学 | A kind of expression of activated goldfish Tgf2 transposons restructuring transposase albumen |
| CN105018523B (en) * | 2015-04-09 | 2019-05-07 | 扬州大学 | A kind of ZB Transposon System and its gene transfer method of mediation |
| AU2016325384B2 (en) * | 2015-09-22 | 2021-07-22 | Julius-Maximilians-Universitat Wurzburg | A method for high level and stable gene transfer in lymphocytes |
| CN105274140A (en) * | 2015-10-28 | 2016-01-27 | 厦门大学 | Method for building muscle cell-specific expression mCherry fluorescent protein zebra fish family |
| CN105409882B (en) * | 2015-12-04 | 2018-03-06 | 上海海洋大学 | A kind of screening carries deletion form Tgf2 transposons goldfish parents to breed the method for high percentage of A-class goods offspring |
| 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 |
| CN108949787A (en) * | 2018-07-05 | 2018-12-07 | 上海海洋大学 | A kind of goldfish Tgf2 transposase and its preparation and store method |
| CN110776570A (en) * | 2019-10-22 | 2020-02-11 | 上海海洋大学 | Recombinant transposase and application thereof |
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| CN101698848A (en) * | 2009-08-26 | 2010-04-28 | 上海海洋大学 | Goldfish autonomous transposon gene gfTol2, transposase coded by gene, and use of gene |
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| CN101698848A (en) * | 2009-08-26 | 2010-04-28 | 上海海洋大学 | Goldfish autonomous transposon gene gfTol2, transposase coded by gene, and use of gene |
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| Akihiro Urasaki等.Functional Dissection of the Tol2 Transposable Element Identified the Minimal cis-Sequence and a Highly Repetitive Sequence in the Subterminal Region Essential for Transposition.《Genetics》.2006,第174卷639-649. * |
| Igor Kondrychyn等.Genome-wide analysis of Tol2 transposon reintegration in zebrafish.《BMC Genomics》.2009,第10卷418/1-13. * |
| Koichi Kawakami.Tol2: a versatile gene transfer vector in vertebrates.《Genome Biology》.2007,第8卷S7/1-10. * |
| 付光明等.青鳉Tol 2 转座子研究进展.《遗传》.2006,第28卷(第7期),899-905. * |
| 邹曙明等.Carassius auratus transposon Tgf2 putative transposase ORF1,putative transposase ORF2, putative transposase ORF3, and putative transposase ORF4 genes, complete cds.《GenBank:HM146132.1》.2010, * |
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