CN103173491A - Application of fusion protein NLS-I-SceI (Nuclear Localization Sequence-I-endonuclease) in preparing reagent for mediating mammal gene transfer - Google Patents

Abstract

The invention discloses an application of a fusion protein NLS-I-SceI (Nuclear Localization Sequence-I-endonuclease) comprising a nuclear localization sequence in preparing a reagent for mediating mammal gene transfer. The amino acid sequence of the fusion protein NLS-I-SceI is shown as SEQIDNO.1 and consists of the nuclear localization sequence NLS and endonuclease I-SceI. The NLS is located at the terminal N of the I-SceI. The fusion protein can specifically identify I-SceI recognition site in a mammal cell to downcut target gene segments containing reverse I-SceI recognition sites at both ends from plasmid to form an NLS-I-SceI-target gene segment complex. Then, the complex is transferred from cytoplasm to nucleus and integrated to genome of a recipient cell so as to obtain a transgenic cell. The fusion protein NSL-I-SceI disclosed is used as the reagent for preparing transgenetic mammal, and has the advantages of high biological safety, convenience, high efficiency and wide application range.

Description

The application of fusion rotein NLS-I-SceI in the reagent of preparation mediate mammalian transgenosis

Technical field

The invention belongs to biological technical field, relate to the application of mRNA in the reagent of preparation mediate mammalian transgenosis of fusion rotein NLS-I-SceI and coding thereof.

Background technology

The mammalian genes transfer techniques is the mammalian genes group to be carried out the important means of genetic modification, initiative medical science transgenic animal model and cultivation transgenic animal new variety.The major technique of existing initiative transgene mammal has: embryonic cell pronuclear microinjection, the transgene clone based on body-cell neucleus transplanting (SCNT), recombining virus carrier infection, transposon-mediated transgenosis and Sperm-mediated transgenosis (SMGT) etc.

The embryonic cell pronuclear microinjection is that the DNA fragmentation that will contain the goal gene sequence imports the fertilised non-human eggs protokaryon, to realize the integration of transgenosis in genome and to obtain thus transgenic animal, be the classical way of cultivating transgene mammal, but this technical efficiency is very low.There are some researches show (Wall, et al. Therionology, 1996,45:57), in the very high mouse of embryonic cell protokaryon sharpness, efficient (embryo number of the transgenic animal number of acquisition/transplanting) by embryonic cell pronuclear microinjection development transgenic mice is only on average 2.6%, and the head that obtains builds in animal the transgenic positive rate lower than 10%.In Mammals, especially large animal (as pig, ox, sheep etc.) beyond mouse, due to embryonic cell protokaryon poor definition, lower based on the transgene efficiency of embryonic cell pronuclear microinjection.For example, utilize the embryonic cell pronuclear microinjection to prepare the efficient of transgenic pig generally lower than 1%(Hammer RE et al. Nature, 1985, be 315:680), only 0.3%(Matrin et al. Transgenic Research in the part report, 2005,14:373), and head builds in pig the transgenic positive rate generally lower than 5%.Because large animal purchase, raising and experimental cost are expensive, the poor efficiency of this technology has seriously restricted the mass-producing preparation of transgenosis large animal.

In order to overcome the deficiency of embryo's pronuclear microinjection technology, the transgene clone technology has been widely used in the preparation of transgene mammal, especially large animal.The transgene clone technology be the transgenic cell that contains goal gene be donor cell, by body-cell neucleus transplanting, transgenosis donor cell core is imported in non-nucleus egg mother cell, build thus and contain genetically modified clone embryos, then grow acquisition transgenosis individuality by clone embryos.This technology can realize in theory that due to take transgenic cell as donor cell head builds the transgenic positive rate of animal 100%.But, because the clone embryos developmental rate is low, cause transgene clone efficient (the clone embryos number of the transgenic and cloned animal number of acquisition/transplanting) very low (generally lower than 3%) (Dai et al. Nature Biotechnology; Lai et al. Science, 2002,295:1089; Kues et al. Trends Biotechnology, 2004,22:286).Secondly, because cloned animal often heteroplasia can occur, cause the transgenic and cloned animal perinatal mortality and die young rate all higher (can up to 40%).Again, because the preparation of transgenosis donor cell needs the drug resistance screening, cause containing drug resistance gene in the genome of the transgenic and cloned animal that obtains, have Biosafety hidden danger, limited the range of application (as food, biomaterial and goods etc.) of transgenic animal.In addition; the transgene clone Technology Need gathers the animal ovocyte on a large scale; need a large amount of, stable ovary source; therefore this technology can only conventional be applied to carry out for a long time the economic class animal (as pig, ox, sheep etc.) that mass-producing is butchered, and is not suitable for non-economy quasi-mode animal (as dog, monkey etc.).

It is efficient transgenic animal technology of preparing that recombinant viral vector (being mainly the lentiviral vectors based on the transformation of the reverse transcription virus gene groups such as HIV-1) infects, its transgenic animal preparation efficiency can reach 14-31%, head builds the Animal Transgenic positive rate can reach 60-95% (Lois et al. Science, 2002,295:868; Whitelaw et al. FEBS Letter, 2004,571:233; Hoffman et al. EMBO Reports, 2003,4:1054), but this technology still has many defectives: at first, the recombinant virus particle suspension of the high titre of this Technology Need, and the packing of infectious titer, concentrated and purifying are complicated, loaded down with trivial details biological procedureses, and the requirement of Biosafety degree is high; Secondly, recombinant viral vector stems from pathogenic viral genome (as HIV-1), there is Biosafety hidden danger, has seriously limited the application of transgenic animal.

Transposon-mediated transgenic technology is the method that effectively prepares transgenic animal, but this Technology Need adds the transposon element in transgene carrier, still possess mobility after causing transgenosis to be integrated into Animal genome, still have the Biosafety hidden danger of similar virus vector.

The Sperm-mediated gene transfer technique is considered to easy and large animal transgenic technology cheaply, but due to this technology poor repeatability, highly unstable, causes it not become at present the routine techniques of preparation transgene mammal.

Summary of the invention

In view of this, the object of the present invention is to provide the application of a kind of fusion rotein NLS-I-SceI, this fusion rotein can specific recognition I-SceI recognition site, and the target gene fragment that two ends is contained reverse I-SceI recognition site is downcut; Two of purpose of the present invention is to provide the application of the mRNA of the above-mentioned fusion rotein NLS-I-SceI of coding.

For achieving the above object, the invention provides following technical scheme:

1. contain the application of fusion rotein NLS-I-SceI in the reagent of preparation mediate mammalian transgenosis of nuclear localization sequence NLS, the aminoacid sequence of described fusion rotein NLS-I-SceI is as shown in SEQ ID NO.2.

Preferably, the nucleotide sequence of encoding said fusion protein NLS-I-SceI is as shown in SEQ ID NO.5.

Preferred, described Mammals is pig or mouse.

2. the application of the mRNA of the described fusion rotein NLS-I-SceI of coding claim 1 in the reagent of preparation mediate mammalian transgenosis.

Beneficial effect of the present invention is: the invention discloses and will contain the reagent of the fusion rotein NLS-I-SceI of nuclear localization sequence NLS for the preparation of the mediate mammalian transgenosis, can be used for the mediate mammalian transgenosis, the preparation transgene mammal, and have following advantage:

(1) biological safety is high, compare with the virus vector transgenic lines transposon carrier system of unifying, present technique only needs to add the recognition sequence of macronucleus enzyme I-SceI in transgene carrier, need not to add the sequences such as transposon or virus vector, be integrated into the transgenosis copy of Animal genome without mobility; Compare with the transgene clone technology, present technique need not to add drug resistance gene in transgene carrier, in the genome of the transgenic animal of acquisition also without drug resistance gene.

(2) easier, present technique only needs the target gene sequence that reverse I-SceI recognition site is contained at mRNA and the two ends of fusion rotein NLS-I-SceI or encoding fusion protein NLS-I-SceI is imported simultaneously the kytoplasm of mammal embryo cell, can effectively realize transgenosis, obtain transgenic animal; Compare steps such as need not loaded down with trivial details cell in vitro cultivation, transgenic cell screening and large-scale nuclear transplantation with the transgene clone technology; System compares with the virus vector transgenosis, has saved the loaded down with trivial details steps such as viral packing, concentrated and purifying.

(3) possesses stable high-level efficiency, the technology of utilizing the present invention to set up, the transgenic positive rate that head builds animal is stabilized in more than 50%, the cultivation efficient of transgenic animal (transgenic animal number/transplanting embryo number) is stabilized in more than 8%, and several times are higher than embryonic cell pronuclear microinjection technology and transgene clone technology; Present technique prepares the stabilised efficiency of transgenic animal, can conventional be used for transgenic animal and cultivate, effectively overcome the Sperm-mediated gene transfer technique existing highly unstable, can not conventionally be used for the shortcoming that transgenic animal are cultivated.

(4) kind is applied widely, and present technique only needs macronucleus enzyme I-SceI molecule and supporting transgene carrier thereof are imported the kytoplasm of mammal embryo cell simultaneously, and need not to import the embryonic cell protokaryon, gets final product the high effect culture transgenic animal; Therefore, compare with embryonic cell pronuclear microinjection technology, do not rely on apparent embryonic cell protokaryon, be applicable to various animal species, the especially low large animal of embryonic cell protokaryon sharpness; Compare with the transgene clone technology, need not to gather a large amount of ovocytes and be used for nuclear transplantation, be applicable to gather on a large scale from the slaughterhouse ovary to obtain the non-economy class Mammals (as non-human primate, dog class etc.) of ovocyte.

Description of drawings

In order to make purpose of the present invention, technical scheme and beneficial effect clearer, the invention provides following accompanying drawing and describe: Fig. 1 is the transgene carrier p2Is structure iron supporting with the NLS-I-SceI molecule.

Fig. 2 is fusion molecule NLS-I-SceI structural representation.

Fig. 3 is NLS-I-SceI and the I-SceI mRNA electrophorogram (1: the I-SceI mRNA after tailing of in-vitro transcription; 2: the NLS-I-SceI mRNA after tailing; 3: the I-SceI mRNA of tailing not; 4: the NLS-I-SceI mRNA of tailing not).

Fig. 4 will encode after the in-vitro transcription mRNA of NLS-I-SceI and I-SceI and transgenosis plasmid p2Is-UBC-eGFP mixing solutions import mouse 1-cell phase embryo kytoplasm, and after injection, 5d observes expression (A:NLS-I-SceI mRNA and the p2Is-UBC-eGFP plasmid hybrid injection group of eGFP gene in mouse embryo cell; B:I-SceI mRNA and p2Is-UBC-eGFP plasmid hybrid injection group).

Fig. 5 is for shifting the eGFP transgenic mice living body fluorescent detected result figure of development by macronucleus enzyme molecule NLS-I-SceI mediated gene.

Fig. 6 builds mouse PCR detected result figure (M:DNA marker for the eGFP transgenosis head that shifts development by macronucleus enzyme molecule NLS-I-SceI mediated gene; 1: negative control; 2-9: head builds the mouse gene group DNA sample; 10: blank (not adding template DNA)).

Fig. 7 is part pig body early embryo figure.

Fig. 8 is the NLS-I-SceI molecule mediates transgenosis in pig body early embryo cell efficiency assessment (wherein blastaea 1-4 is the hyperfluorescenceCeng Yongminggaoyingguang positive, and blastaea 5-6 is the hypofluorescence positive)

Fig. 9 is the living body fluorescent detection that transgenosis head builds pig.

Figure 10 is PCR screening (the M:DNA marker that transgenosis head builds pig; 1-4: the head of birth builds the pig sample; 5: positive sample (ratio with 4pg plasmid/mg genomic dna in contrast pig genomic dna sample adds the transgenosis plasmid); 6: negative control (contrast pig genomic dna is namely only injected the pig sample that the p2Is-UBC-eGFP plasmid obtains); 7: blank (not adding template DNA))

Figure 11 is the schematic diagram that the numerator mediated mammalian genes of NLS-I-SceI shifts.

Embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.

Embodiment 1

One, with the structure of the supporting general transgene carrier p2Is of NLS-I-SceI molecule

1, the following sequence of synthetic, structure as shown in Figure 1, sequence is:

5 '- CgtctcCgggatagttacgctagggataacagggtaatatagttaattaagatctctcgaga attcggtacctctagagtcgacccgggatccatatggttaacgcggccgcatcgat aagcttgggccctggccactgcaggcgcgccgctagccgcggccatggtgtacacc tcagcttaagtttaaactgatcactatattaccctgttatccctagcgtaact GctcttcCgctt-3 ' (SEQ ID NO.1), in this sequence, black matrix is the recognition site of macronucleus enzyme I-SceI, and insert the multiple clone site that forms by a plurality of restriction enzyme enzyme recognition sites between two reverse I-SceI recognition sites, with the sequence subclone of synthetic to the pUC18 plasmid (available from Sigma-Aldrich company, Cat#:D4154) in, BsmBI and SapI restriction enzyme site, namely get the transgene carrier p2Is supporting with the NLS-I-SceI molecule.

Two, the in-vitro transcription Vector construction that contains the NLS-I-SceI molecule

For the macronucleus enzyme I-SceI that makes expression can be positioned in mammiferous nucleus, add mammal nuclear positioning sequence (NLS) at the N of macronucleus enzyme I-SceI end, formation contains the fusion molecule of NLS sequence, called after NLS-I-SceI, its structure as shown in Figure 2, aminoacid sequence is as shown in SEQ ID NO.2.In the present embodiment, the aminoacid sequence of macronucleus enzyme I-SceI is as shown in SEQ ID NO.3, and the amino acid of mammal nuclear positioning sequence is as shown in SEQ ID NO.4.For target animal realized the optimization of codon proneness, side by side except the NLS-I-SceI of potential shearing site, the nucleotide sequence of its coding NLS-I-SceI is as shown in the SEQ ID NO.5.By the synthetic sequence as shown in SEQ ID NO.5 of full gene, and (available from Promega company, T7 promotor downstream Cat#::E1731) obtains the outer transcription vector pT7-NLS-I-SceI of NLS-I-SceI genosome to be cloned into the PCI carrier.The nucleotide sequence of synthetic coding macronucleus enzyme I-SceI simultaneously as shown in SEQ ID NO.6, and is cloned into the T7 promotor downstream of PCI carrier, obtains the outer transcription vector pT7-I-SceI of I-SceI genosome, with in contrast.

Three, by in-vitro transcription obtain respectively the to encode mRNA of NLS-I-SceI and I-SceI

Utilize the ClaI restriction endonuclease abundant enzyme that spends the night respectively to cut in-vitro transcription carrier pT7-NLS-I-SceI and pT7-I-SceI, obtain linearizing template.The enzyme system of cutting is: template plasmid 10 μ g, ClaI restriction endonuclease 20IU, enzyme cutting buffering liquid 5 μ L, and adding afterwards deionized water to final volume is 50 μ L, 37 ℃ are incubated overnight.

After endonuclease reaction finishes, according to following steps, linearizing template is carried out purifying with concentrated:

1) adding Proteinase K (PK) to final concentration in the endonuclease reaction system is 100 μ g/mL, and SDS is to final concentration 0.5%(quality volume fraction), hatch 45min for 50 ℃.

2) sodium acetate soln that adds afterwards, 1/10th volumes in the endonuclease reaction system.

3) add isopyknic phenol in the endonuclease reaction system: the chloroform mixed solution, fully shake mixing, then the centrifugal 10min of 12000g room temperature forwards supernatant in new RNase-free pipe.

4) add the dehydrated alcohol that is equivalent to 2.5 times of volumes of supernatant liquor volume in supernatant ,-80 ℃ of freezing 30min.

5) then at 4 ℃, the centrifugal 10min of 12000g, supernatant discarded is carefully washed tube wall with 75% ethanol of RNase-free, air drying 15min or to 4 ℃ of dry 2h.

6) add the Nuclease-free water dissolving nucleic acid precipitation of 8 μ L.

7) get 1 μ L and carry out nucleic acid quantification, and the template plasmid solution after with purifying divides in the 0.1mL centrifuge tube that is filled to RNAase-free according to 1 μ g/ pipe, is placed in-20 ℃ and saves backup.

In-vitro transcription:

With above-mentioned purifying, concentrated linearizing template, utilize the in-vitro transcription test kit mMESSAGE mMACHINET7 Ultra Kit(Cat#:AM1345 of Ambion company) carry out the tailing of in-vitro transcription and mRNA, this test kit specification sheets is seen in operation in detail.Transcribe complete after, mRNA is dissolved in the TE solution of RNase-free, be divided in the 0.1mL centrifuge tube of RNase-free with the amount of 2 μ L/ pipes, be placed in-80 ℃ of preservations.Simultaneously transcription product is carried out agarose gel electrophoresis, result as shown in Figure 3.

Four, with the structure of supporting enhanced green fluorescence protein (eGFP) the transgene carrier p2Is-UBC-eGFP of NLS-I-SceI molecule

1, the plasmid FUGW(Addgene company that contains the eGFP encoding gene by the PacI+EcoRI double digestion, Cat.#:14883), reclaiming size is the fragment of 2.02kb, this fragment comprises people UBC promotor and eGFP encoding sequence.

2, use the supporting general transgene carrier plasmid p2Is of PacI+EcoRI double digestion and macronucleus enzyme I-SceI, its structure reclaims linearization plasmid as shown in Figure 3.

3, utilize the T4 DNA ligase, the fragment that will contain people UBC promotor and eGFP encoding sequence is connected with linearizing p2Is plasmid, extracts recombinant plasmid 1 after the transformed competence colibacillus bacterium.

4, with EcoRI+BamHI double digestion PCI vector plasmid (Promega company, Cat#::E1731), reclaiming size is the fragment of 280bp, this fragment is SV40 poly(A) sequence; The recombinant plasmid 1 that obtains with EcoRI+BamHI double digestion step 3 simultaneously.

5, SV40 poly (A) sequence is connected with linearizing recombinant plasmid 1 use T4 DNA ligase, the recombinant plasmid 2 of acquisition is the eGFP transgene carrier p2Is-UBC-eGFP supporting with NLS-I-SceI.

6, the transgene carrier carrier p2Is-UBC-eGFP that obtains is carried out purifying with concentrated, and the plasmid after concentrating is placed in-80 ℃ after packing according to 2 μ L/ pipes and saves backup.

Five, the validity of assessment NLS-I-SceI mediate mammalian transgenosis take mouse embryo cell as model cell

Obtaining of mice embryonic: choose the female mouse of sexual maturity, utilize respectively hormone PMSG and HCG to carry out superovulation to female mouse; Mating rear the next morning with the male mouse of sexual maturity chooses the female mouse of the super row of cloudy bolt is arranged; Put to death female mouse by the neck dislocation method, utilize ordinary method clip uterine tube and obtain mouse 1-cell embryo.Manipulating Mouse Embryo Manipulation Manual(Hogan et al. Cold Spring Harbor Laboratory Press, 1994, Second Edition are seen in operation in detail).

The coding NLS-I-SceI of in-vitro transcription or the mRNA of I-SceI and the p2Is-UBC-eGFP transgenosis plasmid of purifying are added respectively in 20 μ L RNase-free ultrapure waters, obtain two kinds of component final concentrations and be the mRNA of 25ng/ μ L and the mixing solutions of plasmid.Then utilize microinjection instrument, the kytoplasm of the mixing solutions of preparing being poured into mouse 1-cell phase embryo (operates in detail and sees Hogan et al. Manipulating Mouse Embryo Manipulation Manual, Cold Spring Harbor Laboratory Press, 1994, Second Edition).Embryo after injecting again be placed in be coated with paraffin oil, the abundant M16 drop of incubation, and at 37 ℃, 5% CO ₂, saturated humidity condition under cultivate.5d after injection observes by the fluorescence inverted microscope, and result as shown in Figure 4.As seen from Figure 4; eGFP only expresses in the injection group embryo that NLS-I-SceI mixes with the p2Is-UBC-eGFP plasmid; and nothing is expressed in the injection group embryo that I-SceI mixes with the p2Is-UBC-eGFP plasmid; show that the NLS-I-SceI molecule can effectively promote gene to shift in mouse embryo cell, and without the I-SceI molecule of NLS signal sequence without this effect.

Six, shift the preparation transgenic mice by the NLS-I-SceI mediated gene

According to above-mentioned steps, mouse is carried out superovulation and obtains mouse 1-cell phase embryo.Then the NLS-I-SceI mRNA and the p2Is-UBC-eGFP plasmid mixing solutions that final concentration are respectively 25ng/ μ L import mouse 1-cell phase embryo's kytoplasm by microinjection, then the embryo after injecting carries out vitro culture.2d after injection, the embryo transfer that is split into 2-cell after injection (is operated referring to Hogan et al. Manipulating Mouse Embryo Manipulation Manual in the female mouse uterine tube of estrus synchronization false pregnancy in detail, Cold Spring Harbor Laboratory Press, 1994, Second Edition).19d after embryo transfer, the female mouse of the false pregnancy of becoming pregnant can be built mouse by output head.By the living body fluorescent of ultraviolet excitation observable transgenic mice, result as shown in Figure 5.Then detecting by PCR can be the positive mouse of DNA level screening transgenic.PCR screening primer is: upstream primer: 5'-actggagaactcggtttgtcgt-3'(SEQ ID NO.7); The downstream primer sequence: 5'-acggccagaatttagcggac-3'(SEQ ID NO.8), PCR product size is 453bp.The PCR condition is, 94 ℃ of denaturations 2 minutes, and 30 seconds, 72 ℃ of 30 seconds, 52 ℃ annealing of 94 ℃ of sex change were extended 1 minute, extended 5 minutes after last 72 ℃.Amplified production is carried out agarose gel electrophoresis, and result as shown in Figure 6.

Experimental result: 113 pieces of present case co-transplantation embryos, 3 of transplant recipients; Become pregnant 2 in the female mouse of acceptor, young 17 of common property; Filter out altogether transgenic positive head by PCR and build 10 of mouse, wherein living body fluorescent is positive 7; The transgenic mice preparation efficiency is 8.8% for (transgenosis head builds mouse/transplanting embryo number), and transgenosis head builds that in mouse, positive rate is 58.8%.

Embodiment 2

One, the validity of assessment NLS-I-SceI mediate mammalian transgenosis take the pig embryonic cell as model cell

Choose the sexual maturity sow, after it is oestrused 17d PMSG Injection hormone 2000IU and after PMSG Injection hormone 72h HCG injection hormone 2000IU, to realize its superovulation.In the 2d morning after oestrus of sow, optional ripe boar once breeds with the pig that oestruses, and selects other end sexual maturity boar to breed for the second time with it in that afternoon.24-36h after first service, the pig body early embryo is obtained in operation, and concrete steps are as follows:

1) by auricular vein implantation quality mark be 3% Sodital sodium solution 10-15mL, realize the sow general anesthesia, and sow is bound on the V-arrangement operating table;

2) conventionally clean, after sterilization sow belly, along the belly median line, between last and the 3rd pair of nipple, hara kiri skin, manadesma, muscle and peritonaeum, size incision are 5-8cm sow;

3) take out sow ovary, uterine tube and part uterus, visible sow ovary this moment surface ovulation point shows that sow ovulates;

4) with internal diameter be the grass tube of 4-6mm two ends passivation, section inserts uterine tube by fimbriae tubae;

5) extract in 38 ℃ of water-baths fully approximately 20mL of the embryo washing water (PBS+1% foetal calf serum) of incubation with syringe, the syringe that will contain embryo washing water by intravenous infusion needle is connected with fallopian tube lumen (it contains syringe needle one end and inserts fallopian tube lumen by the joint portion in uterine tube and uterus, and the other end is connected with syringe);

6) by syringe, embryo washing water is injected uterine tube, and the embryo washing water of collecting the uterine tube of flowing through, flowing out from the grass tube that inserts fimbriae tubae section with the 50mL centrifuge tube of sterilization;

7) embryo washing water of collecting is changed in the sterile petri dish that diameter is 9cm, pick the embryo under stereoscopic microscope, the embryo that pick this moment is generally 1-cell or 2-cell phase, as shown in Figure 7;

8) the pig embryo who picks is placed in cover paraffin oil, incubator fully the nutrient solution drop of incubation balance cultivate (present case pig embryo medium used is PZM-3);

Carry out microinjection according to the described method of embodiment 1, final concentration is respectively the pig body early embryo kytoplasm (annotate: the NLS-I-SceI molecule that the implementation case is used and transgene carrier p2Is-UBC-eGFP are with embodiment 1) that the mixing solutions of the NLS-I-SceI mRNA of 25ng/ μ L and p2Is-UBC-eGFP transgene carrier plasmid imports.Pig embryo after injection is placed in the PZM-3 drop continues to cultivate, and the 5d after cultivation, observe the expression of eGFP in the pig embryo by inverted fluorescence microscope, result is as shown in Figure 8.As shown in Figure 8, visible most of blastaea is the fluorescence positive, has 6 pieces of well-developed blastaeas, and wherein 4 pieces are the hyperfluorescenceCeng Yongminggaoyingguang positive, 2 pieces and are the hypofluorescence positive.

Two, prepare transgenic pig by the numerator mediated transgenosis of NLS-I-SceI

Obtain the pig body early embryo according to above-mentioned case study on implementation, the mRNA of the NLS-I-SceI albumen of then encoding and transgenosis plasmid p2Is-UBC-eGFP import pig body early embryo kytoplasm, and the embryo after injecting is placed in nutrient solution (PZM-3) drop and cultivates.In 24h, the pig body early embryo after injection is transplanted in the uterine tube of the sexual maturity acceptor sow that is in the state of oestrusing after injection.Concise and to the point step is as follows:

1) according to aforesaid method anesthesia, binding acceptor sow, hara kiri skin, manadesma, muscle and peritonaeum take out ovary, uterine tube and part uterus;

2) will pick up embryo suction pipe (U.S., Agtech company) and be connected with the 1mL syringe, and suck in advance one section air in picking up the embryo suction pipe;

3) under stereoscopic microscope, the embryo by the syringe that connects after with piece injection of 20-30 in nutrient solution sucks and picks up in the embryo suction pipe (noting: will suck one section air before the liquid section of embryo place, and suck one section liquid again before air, with anti-pollution);

The embryo suction pipe of picking up that 4) embryo will be housed inserts acceptor pig uterine tube by umbrella section, and pushing syringe imports the embryo in uterine tube;

5) acceptor pig uterus, uterine tube and ovary are put back in abdomen, peritoneal suture, muscle, manadesma and skin successively, and wound is done routine disinfection process.

The acceptor sow of becoming pregnant can give birth to head and build pig after full-term.Detect by PCR respectively and the positive pig of living body fluorescent observation screening transgenic, PCR primer used is with embodiment 1.Utilize GFP Macroscopy System(Hungary, BLS company) transgenosis head is built pig and carry out the living body fluorescent observation, observational technique is: under the half-light condition, with exciting light (wavelength is 460-495nm) irradiation transgenic pig, utilize simultaneously and allow to observe transgenic pig by wavelength for the spectral filter of 500-515nm, detected result as shown in Figure 9, the PCR detected result is as shown in figure 10.46 pieces of the present embodiment co-transplantation embryos, 2 of co-transplantation acceptor pigs are wherein become pregnant for 1, farrow 4; 4 head build pig and are transgenic positive through the PCR detection, and wherein 3 is that living body fluorescent is positive; Transgene efficiency (transgenosis head builds pig number/transplanting embryo number) is 8.7%, and the positive rate that transgenosis head builds pig is 100%.

In the present invention, the principle that the numerator mediated mammalian genes of NLS-I-SceI shifts as shown in figure 11.As shown in Figure 11, the process of the numerator mediated transgenosis of NLS-I-SceI is, I-SceI recognition site on NLS-I-SceI molecular recognition transgene carrier, and cutting-out target gene, the target gene that downcuts is combined with nuclear localization sequence NLS and is formed complex body, the complex body that forms is transported in nucleus by tenuigenin, then is integrated in the nuclear gene group of host cell, obtains at last transgenic cell.

Explanation is at last, above preferred embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is described in detail by above preferred embodiment, but those skilled in the art are to be understood that, can make various changes to it in the form and details, and not depart from claims limited range of the present invention.

＜110〉Military Medical Univ No.3, P.L.A

＜120〉application of fusion rotein NLS-I-SceI in the reagent of preparation mediate mammalian transgenosis

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Lys Glu Tyr Lys Ser Gln Leu Ile Glu Leu Asn Ile Glu Gln Phe

35 40 45

Glu Ala Gly Ile Gly Leu Ile Leu Gly Asp Ala Tyr Ile Arg Ser

50 55 60

Arg Asp Glu Gly Lys Thr Tyr Cys Met Gln Phe Glu Trp Lys Asn

65 70 75

Lys Ala Tyr Met Asp His Val Cys Leu Leu Tyr Asp Gln Trp Val

80 85 90

Leu Ser Pro Pro His Lys Lys Glu Arg Val Asn His Leu Gly Asn

95 100 105

Leu Val Ile Thr Trp Gly Ala Gln Thr Phe Lys His Gln Ala Phe

110 115 120

Asn Lys Leu Ala Asn Leu Phe Ile Val Asn Asn Lys Lys Thr Ile

125 130 135

Pro Asn Asn Leu Val Glu Asn Tyr Leu Thr Pro Met Ser Leu Ala

140 145 150

Tyr Trp Phe Met Asp Asp Gly Gly Lys Trp Asp Tyr Asn Lys Asn

155 160 165

Ser Thr Asn Lys Ser Ile Val Leu Asn Thr Gln Ser Phe Thr Phe

170 175 180

Glu Glu Val Glu Tyr Leu Val Lys Gly Leu Arg Asn Lys Phe Gln

185 190 195

Leu Asn Cys Tyr Val Lys Ile Asn Lys Asn Lys Pro Ile Ile Tyr

200 205 210

Ile Asp Ser Met Ser Tyr Leu Ile Phe Tyr Asn Leu Ile Lys Pro

215 220 225

Tyr Leu Ile Pro Gln Met Met Tyr Lys Leu Pro Asn Thr Ile Ser

230 235 240

Ser Glu Thr Phe Leu Lys

245 246

<210> 4

<211> 31

<212> PRT

＜213〉artificial sequence

<220>

＜223〉nuclear localization sequence

<400> 4

Met Gly Ser Arg Ser Pro Lys Lys Lys Arg Lys Val Pro Lys Lys

5 10 15

His Ala Ala Pro Pro Lys Lys Lys Arg Lys Val Glu Asp Pro Arg

20 25 30

Phe

31

<210> 5

<211> 831

<212> DNA

＜213〉artificial sequence

<220>

＜223〉NLS-I-SceI nucleotide sequence

<400> 5

atgggcagca gaagccccaa gaagaagaga aaggtgccca agaagcacgc cgcccccccc 60

aagaagaaga gaaaggtgga agaccccaga ttcatgtacc cctacgacgt gcccgactac 120

gccggcatga agaacatcaa gaagaaccag gtgatgaacc tgggccccaa cagcaagctg 180

ctgaaggaat acaagagcca gctgatcgaa ctgaacatcg aacagttcga agccggcatc 240

ggcctgatcc tgggcgacgc ctacatcaga agcagagacg aaggcaagac ctactgcatg 300

cagttcgaat ggaagaacaa ggcctacatg gaccacgtgt gcctgctgta cgaccagtgg 360

gtgctgagcc ccccccacaa gaaggaaaga gtgaaccacc tgggcaacct ggtgatcacc 420

tggggcgccc agaccttcaa gcaccaggcc ttcaacaagc tggccaacct gttcatcgtg 480

aacaacaaga agaccatccc caacaacctg gtggaaaact acctgacccc catgagcctg 540

gcctactggt tcatggacga cggcggcaag tgggactaca acaagaacag caccaacaag 600

agcatcgtgc tgaacaccca gagcttcacc ttcgaagaag tggaatacct ggtgaagggc 660

ctgagaaaca agttccagct gaactgctac gtgaagatca acaagaacaa gcccatcatc 720

tacatcgaca gcatgagcta cctgatcttc tacaacctga tcaagcccta cctgatcccc 780

cagatgatgt acaagctgcc caacaccatc agcagcgaaa ccttcctgaa g 831

<210> 6

<211> 705

<212> DNA

＜213〉artificial sequence

<220>

＜223〉I-SceI nucleotide sequence

<400> 6

atgaagaaca tcaagaagaa ccaggtgatg aacctgggcc ccaacagcaa gctgctgaag 60

gaatacaaga gccagctgat cgaactgaac atcgaacagt tcgaagccgg catcggcctg 120

atcctgggcg acgcctacat cagaagcaga gacgaaggca agacctactg catgcagttc 180

gaatggaaga acaaggccta catggaccac gtgtgcctgc tgtacgacca gtgggtgctg 240

agcccccccc acaagaagga aagagtgaac cacctgggca acctggtgat cacctggggc 300

gcccagacct tcaagcacca ggccttcaac aagctggcca acctgttcat cgtgaacaac 360

aagaagacca tccccaacaa cctggtggaa aactacctga cccccatgag cctggcctac 420

tggttcatgg acgacggcgg caagtgggac tacaacaaga acagcaccaa caagagcatc 480

gtgctgaaca cccagagctt caccttcgaa gaagtggaat acctggtgaa gggcctgaga 540

aacaagttcc agctgaactg ctacgtgaag atcaacaaga acaagcccat catctacatc 600

gacagcatga gctacctgat cttctacaac ctgatcaagc cctacctgat cccccagatg 660

atgtacaagc tgcccaacac catcagcagc gaaaccttcc tgaag 705

<210> 7

<211> 22

<212> DNA

＜213〉artificial sequence

<220>

＜223〉PCR detects upstream primer

<400> 7

actggagaac tcggtttgtc gt 22

<210> 8

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉PCR detects downstream primer

<400> 8

acggccagaa tttagcggac

Claims (4)

1. contain the application of fusion rotein NLS-I-SceI in the reagent of preparation mediate mammalian transgenosis of nuclear localization sequence NLS, the aminoacid sequence of described fusion rotein NLS-I-SceI is as shown in SEQ ID NO.2.

2. application according to claim 1 is characterized in that: the nucleotide sequence of encoding said fusion protein NLS-I-SceI is as shown in SEQ ID NO.5.

3. application according to claim 1 and 2 is characterized in that: described Mammals is pig or mouse.

4. the application of the mRNA of the described fusion rotein NLS-I-SceI of coding claim 1 in the reagent of preparation mediate mammalian transgenosis.

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