CN1400310A

CN1400310A - Method for transferring exogenous gene into animal cell

Info

Publication number: CN1400310A
Application number: CN 01120685
Authority: CN
Inventors: 张靖溥; 李光三; 杜淼
Original assignee: Institute of Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2001-07-27
Filing date: 2001-07-27
Publication date: 2003-03-05
Anticipated expiration: 2021-07-27
Also published as: CN1137996C

Abstract

The method for transferring exogenous gene into animal cell includes the following steps: using target gene to construct target gene expression vector, using nuclear localization protein gene to construct nuclear localization protein gene expression vector and transferring the obtained target gene expression vector and nuclear localization protein gene expression vector into the cytoplasm of cultured cell or cytoplasm of animal cylula by means of cotransfection or co-injection or electroporation.

Description

A kind of in zooblast the method for foreign gene transfer

Technical field

The present invention relates to a kind of in zooblast the method for foreign gene transfer.

Technical background

Transgenosis not only can be used for preparing galactophore biological reactor as a kind of technology platform, and in disease model animal, improvement animal varieties, and, all have crucial meaning from molecular level research ontogeny mechanism.Transgenic animal can produce the pharmaceutical protein and the rare albumen that can't obtain with other means of production as a kind of bio-reactor, and have efficient, low cost and product has more characteristics such as natural sex.Its potential economy and social value have been subjected to the attention of countries in the world.Yet the preparation of carrying out transgenic animal during the last ten years on a large scale is still very difficult.One of major obstacle is that transgenic method becomes its bottleneck.

In the preparation of transgene mammal, mainly contain at present two kinds of methods: zygote protokaryon microinjection and be the introduction method of carrier with the sperm.The former requires the zygote protokaryon to have certain visibility meter (this is very difficult) in considerable animal and the operator has quite high operative technique (being difficult for grasping), and easily causes nuclear damage, causes natality very low, the transgenosis failure; The latter is then unstable, poor repeatability.And the cell transfecting method of vitro culture is stranded in transfection efficiency very lowly always, can only carry out cytological observation experiment, can't carry out other research.Therefore people are attempting another approach always: obtain transgenic animal by tenuigenin injection gene.This approach was once attempted in the eighties and the nineties, and the result is [Mario R.et al.Cell 22:479～488 of failure; Mirzayans R, et al.Mutat Res, 281 (2): 115～22].In recent years along with nuclear localization sequence (nuclear localization signals, NLS) and the research of function, some laboratory attempts are carried the target gene member with NLS and are entered nucleus, this trial has obtained initial success [Collas P, etal.Transgenic Res 1998,7 (4): 303～9; Dean DA, et al.Exp Cell Res, 1999,253 (2): 713～22; Wilson GL, et al.J Biol Chem, 1999,274 (31): 22025～32; Collas P, et al.Transgenic Res 1,996 5 (6): 451～8; Page RL, et al.TransgenicRes 1,995 4 (6): 353～60].Collas P etc. prove in the tenuigenin transgenic experiments of zebra fish ovocyte with a kind of NLS-SV40 T antigen of classics and DNA and form mixture, can improve genetically modified integration rate and enter nuclear DNA amount, increase integration site [Collas P on the chromatin, et al.Transgenic Res 1998,7 (4): 303～9; Collas P, et al.TransgenicRes 1,996 5 (6): 451～8]; Go down to posterity and follow the tracks of to detect, transgenosis in this way, foreign gene enters the ratio of germplasm line and brings up to 43% (P＜0.01) from 14%.Page RL[Page RL, et al.Transgenic Res 1,995 4 (6): 353～60] etc. form complex body with artificial synthetic poly-lysine and DNA member, carry out the tenuigenin injection to have obtained transgenic mice, its integration rate is 12.8%; And be zero at the mice in control group integration rate that tenuigenin is only injected the target gene member.The transgenic mice integration rate that obtains by procaryotic injection is 21.7% (only having injected the target gene member).These presentation of results, nuclear localization sequence are to bring into the foreign gene member nuclear from tenuigenin with the complex body form.

Summary of the invention

The purpose of this invention is to provide a kind of in zooblast the method for foreign gene transfer.

Of the present invention in zooblast the method for foreign gene transfer, comprise and use target gene to make up target gene expression carrier; Use the gene constructed nuclear locating sequence expression vector of nuclear locating sequence; And with the target gene expression carrier that obtains and nuclear locating sequence expression vector cotransfection or altogether injection or electroporation in the tenuigenin of culturing cell or the step in the fertilised non-human eggs tenuigenin.

In the method for the invention, described cell can be the zooblast in cultivating, and also can be the zygote of animal.The vitro culture of zooblast, can carry out according to a conventional method [Si Tuzhenqiang etc. cell cultures. Beijing: world book is published Xi'an company, (first version) .58～84 in 1996].[Hogan B is carried out in the acquisition of zygote according to a conventional method, et al.Manipulating the mouseembryo, A laboratory manual.Cold Spring Harbor, New York:Cold SpringHarbor Laboratory Press, 1986.89～175].

Described nuclear locating sequence expression vector is can be at the gene component (dna form) of eukaryotic cell expression nuclear locating sequence.Its encoding sequence can be the biomass cells native sequences, also can be the gene order of synthetic.Its control region has the function that the regulation and control encoding sequence is expressed at eukaryotic cell, can be virus gene sequence, as CMV promotor, SV40 promotor, also can be the promoter region sequence of nuclear locating sequence gene itself, as the promoter of H2A.z.Described target gene expression carrier is to have the purpose of specific control region or the ceneme or the member of target gene.Can be recombinant vectors or natural ceneme.This carrier can be any carrier that can guide foreign gene to express in zooblast well known in the prior art.

The tenuigenin introductory technique that target gene member of the present invention and animal nuclear locating sequence expression vector cotransport, the target gene scope of application is extensive, can be used for: all animal species of a. eucaryon class, as insects, batrachians, birds, vertebrates etc.; B. the cultured cell in vitro of different tissues, tumour cell; And c. sexual cell such as zygote, sperm etc.The gene lead-in mode can be: reagent transfection, electroporation, microinjection etc., appraise and decide position (signal) protein gene expression carrier and being imported in the tenuigenin as long as contain in the cdna solution that is imported into, present method all can promote the integration of foreign gene on host chromosome.In the method for the invention, nuclear locating sequence is meant that a class has the nuclearity of becoming migration and at localized polypeptide of nuclear area or albumen, be present in various eukaryotes, virus or be artificial synthetic product, as act on hormone, the SV40 large T antigen of nuclear, the poly-lysine of synthetic, histone etc.Histone mainly contains six kinds of H1, H2A, H2B, H3, H4, H2A.z etc., recommends the preferential H2A.z of use as appraising and deciding position (signal) albumen.The histone gene sequence can directly be consulted from GenBank.Now provide the login number of the GenBank of mouse histone gene H2A.z used among the embodiment: MMU 70494, and NM 016750.

Target gene expression carrier (DNA) and nuclear locating sequence expression vector (DNA) are total to injection or cotransfection or electroporation in the recipient cell kytoplasm.Earlier target gene expression carrier and nuclear locating sequence expression vector are used DNA purification kit purifying respectively, again with agarose gel electrophoresis estimation DNA concentration.The dna molecular of target gene expression carrier and nuclear locating sequence expression vector is according to 10: 1～1: 5 molecule number mixed (recommending to use 1: 1 molecule number ratio), transfered cell matter (injection altogether, cotransfection or electroporation mode import).

In the method for the invention, preparation of false pregnancy mouse and embryo transfer be [HoganB according to a conventional method all, et al.Manipulating the mouse embryo, A laboratory manual.Cold SpringHarbor, New York:Cold Spring Harbor Laboratory Press, 1986.89～175].

In the method for the present invention, can carry out the in-vitro screening of transfectional cell after culturing cell is transfected, carry out corresponding research again,, be born for transgenic animal until head as the research of cell levels and nuclear transplantation, embryo transfer.Can carry out exogenous origin gene integrator to head for transgenic animal and detect, by PCR-Southern hybridization carry out (Zhang Jingpu etc. Acta Genetica Sinica, 1999,26:135～141).

Brief Description Of Drawings Fig. 1 .A mouse histone H2A.Z structure iron.■ mouse histone H2A.Z gene extron; Mouse histone H2A.Z gene control region and intron.The structure iron of Fig. 1 .B expression of target gene member ox α-s1-casein-people-G-CSF.ox α-s1-casein 5 ' and 3 ' regulating and controlling sequence; ■ human G-CSF genome exon;

Human G-CSF genome intron; The PCR primer location that → integration detects.Fig. 2: natality relatively.1 procaryotic injection; 2 contain the tenuigenin injection of mH2AZ; The tenuigenin injection of 3 no mH2AZ.The injection concentration of target gene is 40 copy/ovum.Move 19-21 piece/acceptor of check figure average out to mouse.Fig. 3 PCR-Southern hybridization portion is .a as a result: the tenuigenin injection that contains the mH2AZ gene is first for mouse.B: the tenuigenin that does not contain the mH2AZ gene is injected the head of acquisition for mouse.P: positive control, N: negative control, numeral are that the head of two groups of injection treatment numbers for mouse.The expression of Fig. 4 human G-CSF in the mouse mammary gland cell.Western Blotting detects, and one anti-ly is the anti-human G-CSF polyclonal antibody of rabbit, and two anti-ly are goat anti-rabbit igg (H+L)-AP, with the colour developing of NBT/BCIP substrate.N: negative control is the normal female mouse whey (4ul) without the transgenosis injection; P1: positive control is people rh-G-CSF albumen (0.54ug); P2: positive control, for people rh-G-CSF albumen (0.54ug) adds normal female mouse whey (4ul); 12 female mouse whey samples of numeral (4ul).

Embodiment

Our laboratory uses the histone H2AZ (mH2AZ) of mouse as nuclear localization signal, promote the transgenosis member ox α-integration of s1-casein-human G-CSF expression vector on host chromosome, being about to expression of target gene member and mH2AZ genetic expression member carries out fertilized egg cell's matter and injects altogether, utilize the directional migration of nuclear locating sequence, bring the destination gene expression member into protokaryon.This routine result shows that method therefor can be brought the expression of target gene member into and obtains transgenosis in the host cell nuclear and integrate mouse, and this method is easier, practical, be easy to promote, can walk around this bottleneck of procaryotic injection technology and a large amount of damages of avoiding zygote, promote the transgenic animal broad application.Therefore, this transgenosis approach can substitute traditional nuclear injecting method fully.This is that domestic first example utilizes the nuclear locating sequence expression vector successfully to prepare transgenic animal.Materials and methods

(i) reagent and test kit .Prime-a-Gene Labeling System and restriction enzyme are all available from Promega company, α-P ³²-dATP is Beijing inferior brightness biological medicine engineering corporation product; The PCR test kit is available from Japanese TaKaRa Biotechnology (Dalian) company limited; The nylon Hybond membrane is a Hybond company product; People rh-G-CSF standard substance (intestinal bacteria product).The anti-human G-CSF of rabbit is available from PEPRO Tech EC Ltd. (England); Second antibody goat anti-rabbit igg (H+L)-AP is available from Vector Laboratories Inc. (U.S.A); Staining fluid BCIP/NBT is available from Beijing Zhong Shan Bioisystech Co., Ltd; The DNA purification kit is a vast Science and Technology Ltd. product; Other reagent all is homemade analytical pure.

(ii) laboratory animal. the Kunming white mouse.

(iii) gene component and primer. the nuclear locating sequence gene is a mouse H2AZ genetic expression member, all from the screening of mouse genomic library, the GenBank of its cDNA sequence is numbered MMU 70494 and NM 016750 in the coding region of its upstream and downstream control region and H2AZ gene.Ox α-s1-casein-hG-CSF member is by ox α-s1-casein 5 ' controlling element 2.2kb and 3 ' regulating and controlling sequence 2kb and human G-CSF genome sequence 1.5kb [the Zhang J P that is spliced, et al.Biotechnology Letters, 2001 (in press)].Be used for primer sequence that the integration of bovine-α-s1-casein-hG-CSF member detects and be 5 ' TCCTCTGACAAACCCTAC3 ' (GF1) and 5 ' CATGCTGTTCCCTACCAC3 ' (GF2).The design of primers strategy is that upstream primer is positioned at ox α-s1-casein 5 ' promoter zone, and downstream primer is positioned at the goal gene zone, and gained PCR product is crossed over the splicing site of 5 ' upstream sequence and goal gene.

The (iv) preparation of transgenic mice. expression of target gene member ox α-s1-casein-hG-CSF (5.7kb) is downcut (see figure 1) from plasmid vector with restriction enzyme KpnI and SacII, with DNA purification kit purifying DNA fragment, and with agarose gel electrophoresis estimation DNA concentration.H2AZ genetic expression member is handled equally.These two kinds of dna fragmentations were according to 1: 1 molecule number mixed, and carry out mouse fertilized egg tenuigenin by the every ovum of each dna molecular 40 copy/2pL and inject, to move in the false pregnancy parent through the embryo of gene injection again, move 313 pieces on ovum altogether, inject experimental group altogether as tenuigenin; Control group only contains the target gene member ox α-s1-casein-hG-CSF with isoconcentration, and does not contain H2AZ genetic expression member.Move 62 pieces on ovum; The two moves 375 pieces on ovum altogether.The fertilized egg cell checks according to an injection group also injection with the transgenosis member ox α-s1-casein-hG-CSF of isoconcentration, the same document of injecting method [Hogan B, et al.Manipulating the mouse embryo, Alaboratory manual.Cold Spring Harbor, New York:Cold Spring HarborLaboratory Press, 1986.89～175], transplant 220 injection ovum in false parent.

(the v) extraction of genomic dna: get mouse tail point 1.5 cm long, extract genomic dna [Hogan B according to a conventional method, et al.Manipulating the mouse embryo, A laboratorymanual.Cold Spring Harbor, New York:Cold Spring Harbor Laboratory Press, 1986.89～175].Detect quality and the concentration of total DNA with 0.7% agarose gel electrophoresis.Carrying out PCR-Southern analyzes.

(vi) PCR-Southern hybridization. the integration of transgenosis member on mouse chromosome adopts the PCR-Southern hybridizing method to detect.Head with above-mentioned different genes combination and different injecting pathways is a pcr template for mouse gene group DNA, and each group all contains the negative contrast of the normal mouse that does not carry out gene injection, the positive contrast template of transgenosis member dna fragmentation.The PCR reactive system carries out according to the test kit explanation, and each reaction contains upstream primer and each 30pmol of downstream primer, dna profiling 0.1ug.35 circulations: 98 ℃ of 500sec, 58 ℃ of 60sec, 72 ℃ of 60sec, 94 ℃ of 60sec.The PCR product is 480bp.Get 15ul PCR product and carry out 0.8% agarose gel electrophoresis, electrotransfer, carry out conventional Southern hybridization (65 ℃) [Sambrook J, et al.1989.Molecular Cloning.A Laboratory Manual. (2 to the Nylon film ^NdEd) .New York.Cold SpringHarbor Laboratory Press].The PCR fragment of the positive template of hybridization probe is with α-P ³²-dATP random priming mark.

(vii) Western blot method detects the specifically expressing of target gene at mammary gland cell.Collect the milk of 7 days head of childbirth for mouse, each newborn sample is got 50ul, with the long-pending TBS damping fluid of monoploid (137mM NaCl, 20mM Tris, pH7.6) dilution, mixing, 4 ℃ are centrifugal 12,000rpm 20 minutes.Get supernatant (whey) be stored in-20 ℃ stand-by [Zhang Jingpu etc. Acta Genetica Sinica, 1999,26:135～141; Simons J P, et al.Nature, 1987,328:530～532].Westernblotting: get 5ul whey and protein denaturation liquid [Sambrook J, et al.1989.MolecularCloning.A Laboratory Manual. (2 ^NdEd) .New York.Cold Spring HarborLaboratory Press] by 1: 1 mixing, boiled 5 minutes, get 4ul and carry out SDS-PAGE (12%) electrophoresis, simultaneously, with the positive contrast of human G-CSF albumen (intestinal bacteria product) of reorganization, the negative contrast of not genetically modified normal mice breast, handle equally.Behind the electrophoresis, sample electrotransfer on the SDS-PAGE gel to nitrocellulose filter, is spent the night 4 ℃ of sealings with skim-milk-TBS.With the anti-human G-CSF polyclonal antibody of first antibody rabbit (be diluted at 1: 3000 TBS-T solution) and transfer film incubation one hour at room temperature.Behind the TBS-T thorough washing, with film and second antibody goat anti-rabbit igg (H+L)-AP (dilution in 1: 3000) incubation one hour at room temperature.Once more behind the thorough washing, with the colour developing of BCIP/NBT staining fluid.Result and discussion 1) ooplasm is injected and the natality of procaryotic injection compares

With the procaryotic injection is control group, and ooplasm injection is divided into two experimental group (with mH2AZ injection group altogether and only inject the expression of target gene component groups), carries out the microinjection transgenosis, calculates the natality of newborn mouse respectively.Calculation formula: natality (%)=(go out cub and count ÷ transplanting ovum number) * 100.Ooplasm injection group is moved 375 pieces on ovum altogether, and going out cub's number is 133.Natality is 35.47%.Wherein, expression of target gene member and mH2AZ expression vector injection group are altogether moved 313 pieces on ovum, several 111 of birth newborn mouse, and natality is 35.46%; Have only the expression of target gene component groups to move 62 pieces on ovum, 22 pieces of birth newborn mouses, natality is 35.48%.The procaryotic injection group is moved 220 pieces on ovum altogether, and going out cub's number is 25.Natality is 11.5% (Fig. 2).

The above results shows, the natality of ooplasm injection group is more than three times of mouse natality of procaryotic injection, the damage that the tenuigenin injection can significantly reduce zygote is described, increase the natality of transgenosis head for mouse, promptly to a certain extent, can improve the probability that obtains transgenic animal, reduce cost.In tenuigenin injection group, the injection group does not have notable difference (being respectively 35.46% and 35.48%) with the natality of only injecting the target gene component groups altogether, illustrates that the mH2AZ expression vector is to the not influence of mouse natality.2) nuclear locating sequence gene and genetically modified integration rate

In order to verify the promotion exogenous origin gene integrator function of nuclear locating sequence, in tenuigenin injection experiment, made do not add the mH2AZ expression vector tenuigenin injection group as contrast.α-the s1-casein-hG-CSF gene component is in the genomic integration situation of mouse to detect ox with the PCR-Southern hybridizing method, and used PCR primer lays respectively at ox α-s1-casein 5 ' control region and human G-CSF gene regions.The result shows, contains the tenuigenin injection group of mH2AZ expression vector, and integration rate is that (Fig. 3 a) for 58% (29/50); And not containing the control group of mH2AZ expression vector, integration rate is zero (0/22) (Fig. 3 b), but has a mouse once positive in twice duplicate detection, is judged to suspicious.The integration rate of procaryotic injection control group is 17.4%.

The common injection of The above results explanation nuclear locating sequence expression vector and target gene member has the effect that promotes that foreign gene is integrated on host chromosome.Can substitute the procaryotic injection method fully and prepare transgenic animal.[Page RL such as Page, et al.Transgenic Res 19954 (6): 353～60] form complex body with poly-lysine and target gene member dna fragmentation, carry out the tenuigenin injection and obtained transgenic mice, integration rate is 12.8%, and the integration rate that does not have the NLS control group is 0%, and the procaryotic injection contrast is 21.7%.Integration rate except that the injection of its tenuigenin among its result is lower than this paper result, other two with we come to the same thing or similar.Difference is that nuclear localization signal used herein is mammiferous mH2AZ, and enter cell with dna form, infer that the mH2AZ expression vector carries out transient expression earlier, its protein product carries the target gene carrier and enters the nuclear district then, and the NLS that its transgenosis integration rate is a synthetic is more than four times.This may be owing to the easier identification of natural nucleus signal for locating and carry due to the DNA.Its mechanism remains further to be studied.3) target gene in mouse milk expression

Adopt the Western-blotting method to detect to whether containing human G-CSF albumen in the mouse milk.Detected 12 female mouse milk of integration male that obtain by the tenuigenin injection altogether, wherein 9 only with expressing human G-CSF albumen.Expression rate reaches 75%, and the preresearch estimates expression level is at 60-550mg/l whey (Fig. 4).

Illustrate that the transgenosis member that most of transgenic mices carry can express at mammary gland cell.In addition, from The above results as can be known, the human G-CSF molecular weight of albumen of reorganization (about 14kD) is littler than the human G-CSF molecular weight of albumen in the mouse milk (about 20kD), and this is because the former for colibacillary product, is without glycosylated molecular form; And the latter may be because due to modifying in the glycosylated back of having passed through of eukaryotic cell expression (Wu Wutong etc., genetically engineered drug one basis is with clinical. Beijing: People's Health Publisher, 1996.169～171).This also illustrates through the protein structure of mammary gland cell expressed proteins and natural form very approaching, so its product activity and physiological function are also more approaching.In addition, in normal lactic, can also examine the G-CSF albumen of tracer level, this also has report (Calhoun D A, et al.Pediatrics, 2000,105:e7 on international publication; Goldman A S, etal.J Mammary Gland Biol Neoplasia, 1996,1:251～8; Juul S E, et al.PediatrRes, 1999,46:263～268; Kling P J, et al.Pediatr Res, 1998,43:216～221), illustrate that some cytokine exists really in mammiferous milk.

In sum, utilize mammals appraise and decide a gene carry out tenuigenin inject altogether the preparation transgenic animal are a kind of easy, practical technology.This technology not only is easy to grasp and popularizes, and genetically modified integration rate and expression rate are also very considerable.Traditional zygote procaryotic injection method can be replaced fully, the development of transgenic animal association area can be promoted greatly.

Claims

1. the method for a foreign gene transfer in zooblast comprises and uses target gene to make up target gene expression carrier; Use the gene constructed nuclear locating sequence expression vector of nuclear locating sequence; And with the target gene expression carrier that obtains and nuclear locating sequence expression vector cotransfection or altogether injection or electroporation in the tenuigenin of culturing cell or the step in the fertilised non-human eggs tenuigenin.

2. in accordance with the method for claim 1, wherein, described nuclear locating sequence is the poly-lysine of the hormone that acts on nuclear, SV40 large T antigen, synthetic, perhaps histone etc.

3. in accordance with the method for claim 2, wherein, described histone is H1, H2A, H2B, H3, H4 or H2A.z.

4. in accordance with the method for claim 3, wherein, described histone is H2A.z.

5. in accordance with the method for claim 1, wherein, the dna molecular ratio of target gene expression carrier and nuclear locating sequence expression vector is 10: 1～1: 5.

6. in accordance with the method for claim 1, wherein, the dna molecular ratio of target gene expression carrier and nuclear locating sequence expression vector is 1: 1.