CN101445805A - Gene capturing carrier and method for establishment of full genome mutated embryonic stem cell bank - Google Patents

Abstract

The invention relates to a set of capturing carriers capable of full genome capturing and a method for establishment of a mutated embryonic stem cell bank using the set of capturing carriers, which belongs to the field of biotechnology. Four capturing carriers related in the invention can respectively capture normal expression genes, induces and captures unexpressed genes and embryo lethal genes in normal cell, so as to subject the genes of full genome to capturing and gene mutation and to establish the monogenically mutated embryonic stem cell bank that covers the full genome.

Description

Gene capturing carrier and set up the method for full genome mutated embryonic stem cell bank

Technical field

The invention belongs to biological technical field, relate to one group of capturing carrier, and utilize capturing carrier to carry out complete genomic gene trap, set up the method for full genome mutated embryonic stem cell bank.

Background technology

Over 30 years, along with the continuous development of genetic technique, adopt the laboratory mouse that gene function is furtherd investigate and be widely used, these researchs make us to human health and disease darker understanding arranged.Yet these biological and genetic technique development are not still made full use of in biology and the medical research, to obtain breakthrough in these fields.Key issue is to improve mouse gene functional research scale and speed, and the cover standard of formulating is identified those drug targets the most relevant with medical science (gene) to come out these results of study there is a standardized explanation in time.A genetic technique that is easy to carry out scale mouse gene functional research is exactly a gene trap.Adopt this method a synthetic dna fragmentation can be inserted randomly in the native gene in the mouse genome with interference base because of normal transcription, thereby reach the purpose that knocks out this gene.Therefore, making up large-scale mutant gene library (preferably complete deactivation) needs the design special carrier, so that filter out because carrier embeds the mutant that is caused.Also require to design carrier simultaneously and make them not only be confined to detect the gene that those are expressed in embryonic stem cell, also can detect the mutant of those genes of in embryonic stem cell, not expressing or embryonic death gene simultaneously.

Mostly under the situation, a gene capturing carrier structure comprises acceptor splicing site, selectable marker gene, with and the inner reverse transcription virus gene group structure that embeds a polyA signal, such structure can be assembled into virion so that infecting embryo stem cell (Abuin, A., G.M.Hansen, and B.Zambrowicz.2007.Gene trap mutagenesis.Handb ExpPharmacol:129-147).When the carrier embedding occurred in the transcriptional activity zone, marker gene began to be transcribed and accurate translation, and expression just can filter out mutant clone according to marker gene.The formation majority of this gene mutation body is to have caught the donor splicing site of native gene and disturbed transcribing of native gene owing to carrying intravital acceptor splicing site, has disturbed gene transcription thereby also there are some to be because carrier directly embeds the exon of gene.Other has a genoid capturing carrier, its selectable marker gene has its promotor, therefore no matter whether carrier embeds the transcriptional activity zone, its selectable marker gene can be activated and transcribe, but needs to catch the exon that contains polyA of native gene 3 ' end in the transcription to guarantee the normal transcription of selectable marker gene.

A problem in the conventional gene trap technology is can't carry out gene to replace (as the little musculus cdna of gene substitution with the mankind) and introduce point mutation, another outstanding defective exactly can not mutagenesis necessary gene (can cause embryonic death) in embryo development procedure, makes the cell bank the set up full gene in the covering gene group fully.

Summary of the invention

This invention is intended overcoming the problem that exists in the conventional gene trap technology, by the use of uniting of one group of four carrier genes different in the genome is caught.Utilize this cover carrier to set up the embryonic stem cell mutant clone on a large scale, make up the embryonic stem cell library, its heritable variation is at random, has certain variation frequency again, the mutant that can guarantee any one gene in the genome can both find in this library, especially survives non-existent embryonic death gene in the mouse.This embryonic stem cell library can obtain the cell mass of specific mutant cell and the development of single mutant cell, and obtains non-human transgenic animal by transgenic technology.

(1) capturing carrier

The invention provides the carrier system that a cover contains four different capturing carriers, unite use and can catch, guarantee that to greatest extent constructed library comprises the gene mutation body in the cover complete genome group the dissimilar genes in the full genome.Two ends, four carrier left and right sides all each have a long terminal repeat (LTR), thus can start capturing carrier in cell transcribe and will be between two LTR of capturing carrier sequence embed in the genome and cause transgenation.Wherein Trp1 is used to be captured in the gene of transit cell record, and Trp2 and Trp3 are captured in the recipient cell not expressing gene, and Trp4 catches the embryonic death gene.Specify as follows:

Trp1 is the carrier that is used for being captured in the recipient cell open gene.This carrier comprises the marker gene (β geo) of a beta-galactosidase enzymes and neomycin resistance gene fusion, the receptor sequence (SA) before the marker gene, the polyA signal (pA) after the marker gene.Gene capturing carrier is embedded into the native gene in the target cell genome, under the effect of native gene promotor and regulatory factor, transcribe, the acceptor splicing site of selectable marker gene upstream will be caught the donor splicing site of native gene exon, form one and merge transcript mutually with selectable marker gene by the part native gene, selectable marker gene is activated and expresses, and utilizes fusion rotein β geo that the gene of catching is carried out expression analysis (Fig. 2).

Trp2 can be used for catching the gene of expressing or not expressing in the cell.Carrier has comprised phosphoglyceric kinase promotor (PGKp), the selectable marker gene (Neo) of selectable marker gene upstream, donor splicing site (SD) and one section exogenous polynucleotide sequence (LTR) that mutagenesis is arranged that is positioned at the promotor upstream in selectable marker gene downstream.The transcript and expression of promoter regulation selectable marker gene in the recipient cell that transforms wherein, normal montage concerns or causes the normal sequence arrangement displacement of native gene exon, the expression of interference and destruction native gene when polynucleotide sequence interference and the expression of destruction native gene for acceptor.The Trp2 carrier is embedded in the intron of native gene in karyomit(e), transcribe with the RNA montage in, exon in the selectable marker gene arrives with the exon splicing that the native gene end contains the ployA signal, and marker gene is able to correct transcript and expression (Fig. 3).The gene that Trp2 can catch must contain one or more intron.

Trp3 is similar to the effect of Trp2, also can be used for catching the gene of expressing or not expressing in the cell, the difference of the two is that Trp3 has inserted one section IRES by the LoxP double team (internalribosome entry site) sequence between selectable marker gene (Neo) and donor splicing site (SD), embeds the problem (Fig. 4) of some preference native gene 3 ' end to avoid the caused carrier of NMD (Nonsense-mediated messenger RNA decay) effect.

Trp4 can mutagenesis and is caught the embryonic death gene, is for carry out the technology that high-throughput (high throughput) conditional gene knockout (conditional knockout) designs in embryonic stem cell.Contain a box (casette) of being formed by acceptor splicing site (SA), beta-galactosidase enzymes and neomycin resistance gene fusion product (β geo) and polyA signal (pA) in the carrier, this box is inserted in the Moloney Leukemia virus skeleton of a no promotor and enhanser, direction of inserting and viral transcriptional orientation opposite (Fig. 5) (Friedrich et al., 1991, Genes and Development, 5:1513-1523).Because the existence of two locus specificity recombination systems (FLPe_frt and Cre-loxP), its carry coding, contain insert positive-sense strand box, that have the mutagenesis function can at first be formed by the FLPe_frt system inversion noncoding, contain the antisense strand that inserts box, do not have the mutagenesis function, and then become positive-sense strand again through the Cre-loxP system inversion.This system utilizes G418 can carry out the high flux screening of gene trap strain, before the blastocyst microinjection produces the variation mouse mutant is reversed once more then, makes mutant gene recover normal to avoid the phenotype of embryonic death.Can be in the specified phase of its growth or specific tissue after obtaining such mouse making mutagenesis insert box by the specificity recombination system reverses once more and causes genovariation (Fig. 5).

Said gene capturing carrier (as Trp4) also can be applied to embed regulatory factor in the genome randomly, evaluation has the system that induces with inhibit feature in animal, carry out the research of regulating and expressing to catching gene, thunderous handkerchief mycin, tsiklomitsin, moulting hormone, glucocorticosteroid and heavy metal inducible system.These systems place these DNA elements in the promotor or near it, inductive or the inhibition transcription factor distinguishing and be attached on the DNA element also can be transformed in the cell.This transcription factor can be attached on the DNA element specifically, and according to the structure of transcription factor, it can be induced or suppress cell and contain the expression of gene of inserting the DNA element.

(2) make up the full genome mutated embryonic stem cell bank and the assignment of genes gene mapping

Adopt conventional molecular biology method, each element on the capturing carrier is cloned on plasmid vector or the virus vector successively, constitute and infect carrier.Through the method for biochemistry or physics, as methods involvings such as liposome method, chemical transfection, electroporation, virus infection method will build infect plasmid vector or virus vector is converted into embryonic stem cell, embed genome, be used for gene trap.Plasmid vector comprises dna vector, and virus vector comprises that retroviral vector, adenovirus carrier, adenovirus related vector, SV40 for basic carrier, vaccinia virus vector, also can be other type of carrier.

Selectable marker gene in the gene capturing carrier is after gene capturing carrier embeds genome, through the intravital regulating and controlling sequence regulating and expressing of cell.At first by medicine, screened as Xin Meisu, resulting positive cell mono-clonal merges through collecting, and promptly forms mutated embryonic stem cell bank.Mono-clonal is transferred to from culture dish and continues in porous organization's culture plate (as 96 orifice plates) to cultivate.Go out this cultivation platform and begin, these are cloned in and are copied into two parts on the porous culture plate, and a plate is used for storing, and another plate is used for analysis subsequently.

Based on round pcr, utilize the gene capturing carrier sequence that genetic transcription regional sequence in its embedded genome is identified.From the positive embryonic stem cell clone of screening, extract genomic dna, remove digested genomic dna, the genomic dna that has digested is connected with suitable restriction enzyme.With the genomic dna after connecting is template, carries out two-wheeled PCR with amplification vector-genome binding site with the nested primer in the LTR sequence.The PCR product is checked order to determine that carrier embeds point (Fig. 6) in genome, the resulting sequence that checks order is exactly all edge sequence ISST (integration site sequencetag) that carrier embeds point, is and remarkable close IPCR (the inverse polymerase chain reaction) sequence of mouse genome.Adopt BLAST software, the mouse genome sequence of ISST and announcement is compared, determine that each clones its carrier and embed the accurate position of putting in genome, the gene of the clone in the library and its mutagenesis is connected.Mouse genome sequence and gene mapping derive from NCBI or UCSC database.

The present invention is applicable to the genomic extensive genetic analysis of any biology that can carry out cell cultures.Every routine techniques that can adopt carries out that carrier imports or any cell of recombinant retroviral carrier transfection all can be used to make up the library, as zoologizeing the genetic tool of the arbitrary gene in the genome.The varient of each gene in the full genome is contained in this library in theory.Because embryonic stem cell variation clone can be gone into blastocyst by microinjection and then produce the variation mouse in the library, so the transgenic mice strain of all genes in the full genome has also been represented in the library.

Same method also may be used to make up nearly all inhuman transgenic animal (maybe can implement the animal of transgeneic procedure).These non-human transgenic animals may comprise as pig, mouse, rabbit, ox, goat and other multiple genetically modified organism, particularly Mammals.The present invention is specially adapted to ox, sheep, pig and other rodentss such as rat, rabbit, cavy and non-human primate.

The transgenic animal of using library described in the invention and/or carrier to produce can be used to study basic biological procedures and disease incidence mechanism, these diseases include but are not limited to aging, cancer, autoimmune disorder, disease of immune system, alopecia, gland disease, inflammatory diseases, diabetes, sacroiliitis, hypertension, atherosclerosis, cardiovascular disorder, lung disease, nerve or Skeletal system are degenerated, senile dementia, Parkinson's disease, asthma, development obstacle or unusual, Infertility, epithelium ulcer and microorganism disease (Mandell et al., 1990, ＂ Principles and Practice of Infectious Disease ＂ 3rd.ed.).Equally, above-mentioned animal and cell also can be used for the research of functional genomics.

Description of drawings

Fig. 1: four capturing carrier structural representations of Trp series

(Trp1-capturing carrier 1; Trp2-capturing carrier 2; Trp3-capturing carrier 3; Trp4-capturing carrier 4)

Fig. 2: the principle of work (1,2,3 are three exons of genomic gene X) of utilizing the Trp1 carrier

Fig. 3: the principle of work (1,2,3 are three exons of genomic gene X) of utilizing the Trp2 carrier

Fig. 4: the principle of work (1,2,3 are three exons of genomic gene X) of utilizing the Trp3 carrier

Fig. 5: the principle of work (1,2,3 are three exons of genomic gene X) of utilizing the Trp4 carrier

Fig. 6: the identifying mark carrier embeds the strategy based on PCR (1,2,3 is three exons of genomic gene X) of point

Embodiment

(1) structure of capturing carrier

The structure of capturing carrier is according to Protocols in Molecular Biology, through enzyme cut, connect, steps such as screening, purifying, successively each element is cloned among the plasmid pBR322, element accepted or rejected and sorted according to making up required capturing carrier.These elements comprise the resistant gene (β geo) that neomycin resistance gene (neo) or beta-galactosidase enzymes and neomycin resistance gene merge; Long terminal repeat (LTR); Acceptor splicing site (SA); Donor splicing site (SD); Poly (A) sequence; Beta-galactosidase enzymes (LacZ); Phosphoglyceric kinase (PGK) promotor (PGKp); Internal ribosome enters sequence (IRES); FLP recombinase (fliprecombinase) recognition site (frt-F3); Cre/lox recombination system recognition site (LoxP-Lox511) etc.

(2) make up full genome mutated embryonic stem cell bank and embedding point gene sequential analysis location

Embryonic stem cell comes from the embryo-sac cell (Hogan that mouse is a delayed maturity after the C57BL/6N pregnancy, B., R.Beddington, F.Costantini, and E.Lacy.1994.Manipulating the Mouse Embryo:A laboratorymanual.Cold Spring Harbor Laboratory Press).The C57BL/6N cell through confirm based on complete genomic snp analysis (The Jackson Laboratory, Bar Harbor, ME).

The cultivation of embryonic stem cell is operated (Hogan by standard program, B., R.Beddington, F.Costantini, and E.Lacy.1994.Manipulating the Mouse Embryo:A laboratory manual.Cold Spring Harbor LaboratoryPress, Matise, M., W.Auerbach, and A.Joyner.2000.Production of targeted embryonic stem cellclones.In Gene Targeting, A Practical Approach (ed.A.Joyner), 101-132.Oxford UniversityPress), culture condition is 37 ℃ and 5%CO ₂, adopt Dulbecco ' s DMEM and FBS foetal calf serum to cultivate.Embryonic stem cell is at M15 substratum (4.5g/mL D-glucose and sodium bicarbonate and do not contain the DMEM of Pyruvic acid sodium salt; 15%FBS; The 2mM L-glutaminate; The 50U/mL penicillin G; 50 μ g/mL Streptomycin sulphates; 1 * 10 ^-4The M beta-mercaptoethanol) carries out on.The Trypsin of embryonic stem cell turns into carrying out by the following method: handle 15-20mins in the phosphate buffered saline buffer that contains 0.25% trypsinase and 0.04%EDTA (PBS does not contain Ca+ and Mg2+).Embryonic stem cell refrigerates in final concentration and contains in the DMEM solution of 20%FBS and 10%DMSO.

The whole process that embryonic stem cell is cultivated all is to remain on mouse embryo fibroblast feeder cell individual layer, that adopt the ametycin inactivation to handle to carry out (McMahon, A.P.and A.Bradley.1990.The Wnt-1 (int-1) proto-oncogene isrequired for development ofa large region ofthe mouse brain.Cell 62:1073-1085).These mouse embryo fibroblast feeder cell show resistance (carrying the Neo transgenosis) to microbiotic, therefore can adopt the G418 microbiotic to carry out the positive as selective agent and select.In addition, embryonic stem cell is cultivated in the servicing operations, leukaemia inhibitory factor (the Leukemia Inhibitory Factor that should contain about 1000U/ml in its substratum, LIF) to suppress the differentiation of embryonic stem cell, prolong (the Nichols that holds time of its versatility, J., E.P.Evans, and A.G.Smith.1990.Establishment ofgerm-line-competent embryonic stem (ES) cells using differentiation inhibiting activity.Development 110:1341-1348).

About the existing detailed report (Soriano of the method for assembling and titration capturing carrier virus particle, P., G.Friedrich, and P.Lawinger.1991.Promoter interactions in retrovirus vectors introduced into fibroblasts andembryonic stem cells.J Virol 65:2314-2319).The assembling of gene capturing carrier be to use GP+E assembling clone (ATCC, Manassas, VA).For guaranteeing the high titre of the retrovirus capturing carrier that assembling is correct, after using 10 times, virus production cell (VPCs) just must change.Every new cultivate a VPCs, titre will define according among every ml microbiotic being had group's number that the embryonic stem cell of resistance forms.Infected preceding 24 hours, when the virus production cell reached 70% fraction of coverage, the amount of its substratum will suitably reduce (every 10cm ²Last 1ml) being beneficial to virus concentrates.After 24 hours, the suspending nutrient solution centralized collection is also passed through 0.2 μ m screen and is filtered.

It is 10cm, long to have on the Tissue Culture Dish of mouse embryo fibroblast feeder cell that about 200 ten thousand embryonic stem cells are inoculated into diameter, after cultivation in 24 hours, add a certain amount of virus production cell cultures suspension and infect, the amount (concentration of virus particle) of suspension that requirement adds reaches and makes the clone that can produce about 200 anti-G418 on the every 10cm culture dish of diameter.Infect and carried out resistance screening (G418200 μ g/mL) in back 24 hours.After 10 days screening and culturing, mono-clonal is selected to carry out trypsinized to 96 orifice plates and handles and be separated into unicellular.These single-cell suspension liquid are transferred to 96 holes then, contain on the Tissue Culture Dish of individual layer mouse embryo fibroblast feeder cell and cultivate.Cell is again by trypsinized after 3 days, transfers to 2 96 new holes then, contains on the Tissue Culture Dish of individual layer mouse embryo fibroblast feeder cell and cultivate.After cultivation in 3 days, one of them is used for sequencing analysis, another low tempertaure storage.The final sudden change stem cell bank that contains 300,000 embryonic stem cell genovariation strain systems of setting up.

Extracting genome DNA is directly to carry out from the 96 porocyte culture plates that the embryonic stem cell that overgrows with anti-G418 is cloned.Before carrying out DNA extraction, embryonic stem cell can be stored in-80 ℃ earlier with after the PBS rinsing 2 times.Embryonic stem cell adopts lysis buffer (50mM Tris, pH7.5; 50mM EDTA, pH8.0; 100mM NaCl; 1%SDS and 3mg/mLProteinase K) handle, in bathing, 65 ℃ of temperature spend the night then.DNA adopts TE damping fluid resuspending through alcohol wash and post precipitation.

In order to obtain to be used for the dna profiling of inverse PCR (IPCR), genomic dna at first will adopt suitable restriction enzyme, and BamHI and BglII or HincII and MscI carry out enzyme and cut.Enzyme is cut process and is still directly carried out in 96 porocyte culture plates.Add the T4 ligase enzyme immediately after the digestion back adopts high-temperature inactivation to stop endonuclease reaction in 37 ℃ of temperature are bathed and carry out ligation.Use after ligation is finished with gene capturing carrier complementary nest-type PRC primer (forward primer1, reverse primer1, forward primer2, reverse primer2) and carry out two-wheeled PCR reaction with amplification vector-genome binding site.Purifying products carries out (Millipore, Billerica, MA) also order-checking, sequencing primer: primer 1, primer 2 on the MultiScreen in 96 holes PCR purifying plate.The sequencing result of gained utilizes BLAST software that the embedding site flanking sequence of capturing carrier is compared with the mouse genome sequence of announcing recently, determines the exact position of each ISST in the mouse genome.

The present invention is described by preferred embodiment, and person skilled is not obviously breaking away under the prerequisite of the present invention, and the techniques described herein scheme is done suitable conversion, made up and reappear the present invention.Replacement that all are relevant and change are conspicuous to art technology and people, and they all are regarded as being included in the middle of the scope of the present invention.

Sequence table

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Claims (9)

1, one group is used for the capturing carrier group that full genomic gene is caught, and it is characterized in that the structural order of four carriers from 5 ' to 3 ' is respectively:

Trp1：LTR-SA-βgeo-pA-LTR；

Trp2：LTR-SA-LacZ-pA-PGKp-Neo-SD-LTR；

Trp3：LTR-SA-LacZ-pA-PGKp-Neo-LoxP-IRES-LoxP-SD-LTR；

Trp4：LTR-frt-F3-LoxP-Lox511-SA-βgeo-pA-LoxP-Lox511-frt-F3-LTR；

Wherein LTR is a long terminal repeat; SA is an acceptor splicing site; SD is a donor splicing site; Neo is a neomycin resistance gene; β geo is beta-galactosidase enzymes and neomycin resistance gene fusion product; PA is poly (A) sequence; LacZ is a beta-galactosidase enzymes; PGKp refers to phosphoglyceric kinase (PGK) promotor; IRES is that internal ribosome enters sequence; Frt-F3 is FLP recombinase (flip recombinase) recognition site; LoxP-Lox511 is a Cre/lox recombination system recognition site.

2, vehicle group as claimed in claim 1 is characterized in that four carriers unite use.

3, vehicle group as claimed in claim 1 is characterized in that and can catch full genomic gene.

4, vehicle group as claimed in claim 1 is characterized in that especially can catching the embryonic death gene.

5, a kind of method of setting up full genome mutated embryonic stem cell bank is characterized in that comprising step:

A: with the capturing carrier group

Trp1：LTR-SA-βgeo-pA-LTR；

Trp2：LTR-SA-LacZ-pA-PGKp-neo-SD-LTR；

Trp3：LTR-SA-LacZ-pA-PGKp-Neo-LoxP-IRES-LoxP-SD-LTR；

Trp4：LTR-frt-F3-LoxP-Lox511-SA-βgeo-pA-LoxP-Lox511-frt-F3-LTR；

Be built into plasmid vector or retroviral vector;

B: the plasmid vector or the retroviral vector of structure are converted into embryonic stem cell;

C: screen positive embryonic stem cell, determine to catch gene order, full genomic gene mutated embryonic stem cell bank is set up in the position of gene location in genome.

6, method as claimed in claim 5 is characterized in that plasmid vector is among pBR, pUC, pSP or the pGEM.

7, as method as described in the claim 5, it is characterized in that embryonic stem cell is the embryonic stem cell of mouse, rat, rabbit, cavy, pig, sheep or goat.

8, embryonic stem cell bank as claimed in claim 5 is characterized in that this cell bank comprises all genes of full genome.

9, all genes of full genome as claimed in claim 8 is characterized in that comprising the embryonic death gene.

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