CN102747096B - Gene search vector, random gene mutation control method and application thereof - Google Patents
Gene search vector, random gene mutation control method and application thereof Download PDFInfo
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Abstract
The present invention relates to a gene search vector, a random gene mutation control method and application thereof. Specifically, the present invention relates to a vector for gene search system, a random gene control method, a method for screening high transfer human tumor cell lines, a method for screening a gene for improving human tumor cell line transfer, a whole-genome random mutation library obtained by using the random gene control method, pharmaceutical application of the gene obtained by using the method for screening gene for improving human tumor cell line transfer, pharmaceutical application of an inhibitor of the gene, and pharmaceutical application of an inhibitor of an expression product of the gene. The gene obtained by the method of the invention, the inhibitor of the gene, the expression product of the gene and the inhibitor of the expression product of the gene can be applied to development of specific antitumor drug.
Description
Technical field
The invention belongs to random gene mutation method field.Specifically, the pharmaceutical applications of the inhibitor of the pharmaceutical applications of gene, the pharmaceutical applications of the inhibitor of described gene and the protein expression product of described gene that the present invention relates to gene hunting system carrier used, random gene regulate and control method, the method for screening high-transfer human tumor cell line, the method for screening the gene improving human tumor cell line transfer, the full-length genome libraries of random mutants utilizing described random gene regulate and control method to obtain and utilize described method screening of screening the gene that raising human tumor cell line shifts to obtain.
Background technology
Along with completing smoothly of the Human Genome Project, life science field enters the genome times afterwards comprehensively.
Completing of genome sequence determination is only the first step of genome plan, larger challenge will be how to determine gene function and decode whole genetic information
1,2.A lot of genetics technology is used to find the gene controlling cancer metastasis at present, comprise chemomorphosis DNA, retrovirus inserts mutagenesis, small interfering RNA (siRNA), nuclease (ribozymes), gene targeting (Shot-Gun), transgenic animal and biochip technology, these methodological function genomics research methods are widely used in the research of cancer, and the discovery of new gene has been applied to early discovery, the Diagnosis and Treat of malignant tumour
3-6.Genome sequencing analysis display carcinoma of the pancreas recently about carcinoma of the pancreas about has 63 transgenations, relates to 12 crucial signal transduction pathways
7, wherein core path comprises and only has individual gene to suddenly change dominant path, as KRAS signal transduction pathway; The path of several transgenation, as TGF-signal β Signal Transduction Pathways; The path of many transgenations, as regulated the signal transduction pathway of the integrin of invasion and attack, the signal transduction pathway etc. that Guanine Triphosphatase (GTPase) relies on.The imbalance of these key signal Signal Transduction Pathways and frequently transgenation are enough to explain the unique complicated biological characteristics of carcinoma of the pancreas self, and the molecular mechanism also for deeply understanding carcinoma of the pancreas transfer brings certain difficulty.Therefore, transgenation and the transfer tool contacted studying carcinoma of the pancreas between these sudden change and the biological characteristics of its uniqueness play a very important role frequently to determine carcinoma of the pancreas.
Present stage utilizes the method for functional genomics technical study cancer metastasis to mainly contain microarray analysis, RNA interference and insertion mutation etc.Microarray be grow up late 1980s with high-density be feature can be used for the new gene functional research technology of of extensive gene expression detection difference; Cardinal principle utilizes molecular hybridization principle by being printed on the DNA probe of a large amount of known portions sequence and detection sample hybridization, by detecting hybridization signal intensities and data processing, thus comprehensively carries out quantitative analysis to the expression of gene; Its major advantage is high-throughput, can compare the expression of lots of genes
8-10.Utilize the difference of genetic expression between analyzing biochips transitivity and non-metastatic tumour, present stage has the gene that investigator finds some relevant to cancer metastasis.But, the gene that biochip comparative experiments group and control group differential expression obtain is thousands of, for follow-up analysis brings great difficulty, investigator must have very high professional standards and need follow-up functional verification, and the gene obtained is all certain cancers metastasis related gene.In recent years, RNA interference screening tumour occurs and metastasis related gene aspect achieves very much progress, the genes involved of utilize this technology to filter out key gene new on relevant p53 path occurs tumour, the gene relevant to the migration of epithelium tumor cell and the cancer metastasis the first step, liver cancer occur to be correlated with new gene, melanoma metastasis
11-16.But this technology needs the sequence knowing target gene in advance, just can design the dsRNA of this target gene reticent, the scope of genescreen can be reduced like this, often miss some unknown genes; In addition, target gene can only knock out by RNAi technology, cannot certain gene of process LAN, needs with the use of cDNA library technology
17, so also limit its application.Insertion mutation utilizes known foreign DNA insertion sequence as mark, genetic expression and gene product need not be determined in advance, destroy gene structure and cause sudden change, can directly verify Individual genes and screen relation between proterties, be a kind of desirable functional genome's science study method.Have that bibliographical information utilizes this technology screening to occurring with tumour, metastasis related gene at present, but the problem of puzzlement the method application is the efficiency of its insertion mutation, need foreign DNA high effective integration to genome on the one hand, need two allelotrope simultaneous mutations on the other hand
18.
At present, utilizing insertion mutation to carry out the more instrument of functional genomics research application is transposon.Within 1996, studies have reported that the instrument that application retrovirus inserts as gene and the tumor suppressor gene tsg101 made new advances in conjunction with corresponding phenotypic screen
19.Except retrotransponsons, using more has Sleeping Beauty and PiggyBac transposon, and the gene in insertion mutation and marker gene group, then carries out screening mutant clone according to corresponding functional character, be finally separated and clone gene
20-24.Sleeping Beauty is first DNA transposon system-SB transposon being applied to mammalian cell, research is had to confirm compared with retrotransponsons, its swivel base site bias is relatively little, immunogenicity is low, the multinomial advantages such as transposition efficiency is high, can random mutation gene, for gene functional research and disease research all significant
25.PiggyBac is a kind of moveable DNA element separated from moth, and insertion point prefers to the intron region of gene, and its integrating frequency is high, restricted less by organism kind.Within 2005, demonstrate piggyBac transposon system first at the swivel base that can realize efficient stable in mammalian cell
26; And a lot of experiment has confirmed that the ability of the exogenous genetic fragment that PB transposon system carries is strong, the highest 14.3kb of carrying fragment and transposition efficiency can not be affected; Insertion point more (67%) to be positioned near transcription unit or wherein; Transposition efficiency is high; On a complete set of karyomit(e) of cell, all have distribution after swivel base, gene fraction of coverage is high
27-29.In addition, have research and utilization PB system in mouse embryo stem cell, set up mutated library within the scope of full-length genome, the gene fraction of coverage that result shows its library is desirable, is easy to find new gene
30,31.Therefore, the efficient insertion mutation instrument that PiggBac Transposon System is ideal, can play a significant role, and the feature affecting encoding gene makes it in functional genomics research, have outstanding advantage more in the horizontal screening-gene of full-length genome.
Carcinoma of the pancreas is one of the poorest alimentary system malignant tumour of prognosis.In the past in Two decades years, achieve important progress about its generation, development, diagnosis, Therapy study, annual mortality ratio and annual PMR decline to some extent, but still can not improve the long-dated survival time of Pancreas cancer patients.Because the biological characteristics of the special anatomical position of pancreas, highly invasion and attack makes it be difficult to early discovery, clinical medical time mostly be patients with terminal, shift in patient body, Resection Rate is less than 20%, five year survival rate is still less than 5%, and wherein transfer is the main reason of Endodontic failure
32,33.Metastases refers to that tumour cell leaves original site and the complex process of growth in neologism or tissue, it is the process of a multi-step, comprise cancer cells lose Adhering capacity, vasculogenesis, identification of lymphatic and blood vessel invasion, enters circulation, escape the immunosurveillance of body and survive, stop at capillary vessel place, pass vessel wall and enter surrounding tissue, and structures locating growth forms new metastasis a long way off
34.Think that molecular mechanism that it occurs is that the protein of many genes in this complex process and coding thereof has played vital role at present
35,36.Therefore, these genes and the relevant hereditary path thereof of finding out behind will be the breaches solving metastases.
Therefore, need a kind of molecular genetics methods of uniqueness to study the transfer of carcinoma of the pancreas and/or other malignant tumours, this technology directly can determine the cause-effect relationship of transgenation and cancer metastasis, screen the key gene and candidate's passage that control carcinoma of the pancreas and/or other Malignant tumor of bonal metastasis very efficiently, analyse in depth the molecule mechanism of transfer, for the Diagnosis and Treat of carcinoma of the pancreas and/or other malignant tumours lays the foundation.
Summary of the invention
Therefore, technical purpose of the present invention is to seek the transfer of a kind of efficient research carcinoma of the pancreas and/or other malignant tumours, the cause-effect relationship of transgenation and cancer metastasis and high frequency zone and controls carcinoma of the pancreas and/or the key gene of other Malignant tumor of bonal metastasis and the method for candidate's passage, and the gene preparation medicine utilizing described method to screen.
Therefore, a first aspect of the present invention relates to a kind of gene and searches carrier, it comprises tsiklomitsin response element, piggbac transposon, preferably, described tsiklomitsin response element is obtained by two sections of tsiklomitsin response element splicings, and preferably, described gene searches carrier also containing neomycin resistance gene, preferably, described gene searches carrier also containing Plasmid replication origins.
A second aspect of the present invention relates to a kind of random gene regulate and control method, and it comprises step:
1) human tumor cell line is used cag-tTA plasmid transfection, obtain the Tet-off cell strain of stably express activating transcription factor (tTA), preferably, described human tumor cell line is human pancreas cancer cell strain, most preferably, described human tumor cell line is human pancreas cancer cell strain Aspc-1;
2) gene as above is searched the plasmid co-transfection step 1 of carrier and effective expression transposase MPB) the Tet-off cell strain that obtains, obtain full-length genome cell homozygous mutant, preferably, the plasmid of described effective expression transposase is MPB
37.
A third aspect of the present invention relates to a kind of method of screening high-transfer human tumor cell line, and it comprises step:
3) by the step 2 as above of significant quantity) the middle full-length genome cell homozygous mutant inoculation nude mice obtained, preferably, described nude mice is BALB/C nude mice (purchased from Nat'l Pharmaceutical & Biological Products Control Institute);
4) grow tumour in nude mouse to be seeded or put to death mouse after manifesting morbid state, be separated and obtain metastasis and carry out primary culture in vitro, obtain high compared with the human tumor cell line of contrast transfer and/or the human tumor cell line of low transfer, the human tumor cell line of described contrast is human pancreas cancer Aspc-1 cell strain.
A fourth aspect of the present invention relates to a kind of method of screening the gene improving and/or reduce human tumor cell line transfer, and it comprises step:
5) by step 4 as above) in the human tumor cell line of the height transfer that obtains and/or low transfer clone gene by the method for Splinkette PCR and search sequence around carrier, then found the gene of this position by blast comparison.
A fifth aspect of the present invention relates to a kind of full-length genome libraries of random mutants utilizing random gene regulate and control method as described in respect of the second aspect of the invention to obtain.
The gene of the method acquisition that a sixth aspect of the present invention relates to described in a kind of the present invention of utilization the 4th aspect is preparing the purposes in antitumor drug, and preferably, described tumour is carcinoma of the pancreas.
A seventh aspect of the present invention relates to a kind of gene utilizing the method described in the present invention the 4th aspect to obtain, and it is used as anti-tumor drug, and preferably, described tumour is carcinoma of the pancreas.
The purposes in antitumor drug prepared by the inhibitor that a eighth aspect of the present invention relates to the gene that the method described in a kind of the present invention of utilization the 4th aspect obtains, and preferably, described tumour is carcinoma of the pancreas, and preferably, described inhibitor is siRNA.
A ninth aspect of the present invention relates to a kind of inhibitor of the gene utilizing the method described in the present invention the 4th aspect to obtain, and it is used as anti-tumor drug, and preferably, described tumour is carcinoma of the pancreas, and preferably, described inhibitor is siRNA.
The purposes in antitumor drug prepared by the inhibitor that a tenth aspect of the present invention relates to the protein expression product of the gene that the method described in a kind of the present invention of utilization the 4th aspect obtains, preferably, described tumour is carcinoma of the pancreas, preferably, the antibody of the protein expression product of described inhibitor utilizes the method described in the present invention the 4th aspect to obtain gene for specificity is anti-, preferably, described antibody is monoclonal antibody.
Gene constructed by the present invention searches carrier, utilize described gene to search carrier and express the method for the Tet-off cell strain of activating transcription factor (tTA) and the plasmid randomly regulating gene of effective expression transposase MPB, the method of screening high-transfer human tumor cell line, the method that screening improves the gene of human tumor cell line transfer is study the transfer of carcinoma of the pancreas and/or other malignant tumours, directly determine the cause-effect relationship of transgenation and cancer metastasis, screen the key gene and candidate's passage that control carcinoma of the pancreas and/or other Malignant tumor of bonal metastasis efficiently, analyse in depth the molecule mechanism of carcinoma of the pancreas and/or other pernicious treatment transfers, Diagnosis and Treat for carcinoma of the pancreas and/or other malignant tumours lays the foundation and provides a whole set of favourable instrument, the full-length genome libraries of random mutants utilizing the method for described randomly regulating gene to obtain provides the foundation for the gene screening other control objectives shapes.Utilize the gene that aforesaid method of the present invention obtains, the inhibitor of this gene, the inhibitor of the expression product of the expression product of this gene and this gene, the medicine of specificity antineoplastic can be developed.
Accompanying drawing explanation
Fig. 1: the principle showing random gene of the present invention sudden change control technique.Gene searches the replication orgin that carrier (GSV) comprises piggyBac transposon skeleton, two tsiklomitsin response elements, neomycin resistance gene, plasmid.PiggyBac transposon can by the mechanism integration of " shear-paste " to genome under the help of transposase; Tsiklomitsin response element is by the activation of activating transcription factor (tTA), and startup is transcribed; Neomycin resistance gene is as selection markers, and can degrade neomycin drug G418; The replication orgin of plasmid is that plasmid amplification copies requisite element.When gene to search in vector integration to encoding gene and by the activation of tTA after transcriptional orientation contrary with this genetic transcription direction time, the antisense RAN (AntisenseRNA) with this gene transcript complementation can be obtained, thus block the translation (A figure) of this gene; If GSV to be inserted between encoding gene and start transcriptional orientation identical with this gene time, then can strengthen the expression (B figure) of this gene.Tetracyclines derivative (DOX) can induce its conformation to change in conjunction with TtTA, hinders it in conjunction with tsiklomitsin response element, thus can not start and transcribe; Therefore, can realize inserting the expression manual control of gene by adding DOX.
Fig. 2: show the experiment flow figure utilizing random gene sudden change control technique Platform Screening to control the key gene of carcinoma of the pancreas transfer.First set up Tet-off expression regulation system at human pancreas cancer cell strain (Aspc-1) and obtain Tet-off cell strain, then utilize gene to search carrier and set up full-length genome cell homozygous mutation library, in nude mouse, screening obtains high-transfer cell strain, carry out the cause-effect relationship checking of transgenation and function again through Dox, obtain candidate gene finally by molecular cloning and blast comparison.
Fig. 3: A shows the schema building gene search carrier, and it comprises acquisition and the splicing of TATA-14Tet fragment and Neo-PA-Ori fragment, and TATA-14Tet-Neo-PA-Ori fragment inserts the insertion of piggyBac transposon and CMV promoter; B shows the collection of illustrative plates of the gene search carrier that the present invention builds.
Fig. 4: show the expression that luciferase reporter gene detects activating transcription factor (tTA): utilize luciferase reporter gene to detect tTA to the Activation Activity of tsiklomitsin response element and the regulation and control degree by DOX, compared with the Aspc-1 cell strain of contrast, the tTA activity of 2B6,2D2,5A3 tri-clone is higher, and by the regulation and control of DOX more than 500 times.
Fig. 5: the concentration showing GSV and MPB is groped.The high effective integration of GSV needs the help of MPB, in order to obtain enough large full-length genome cell homozygous mutation library, needing the concentration of suitable GSV and MPB, and avoiding the radom insertion of GSV as far as possible.MPB can improve the integration efficiency (A figure) of GSV greatly, in order to reduce GSV radom insertion, the transfection amount of GSV will be reduced as far as possible, but the content of GSV can affect the follow-up mutant clon number obtained very little, therefore need to consider and decide GSV consumption, along with the increase of GSV, the colony counts that obtains also increases (B figure) gradually, the ratio of GSV and MPB also determines the number of obtained mutant clon, bibliographical information is had to there will be extra-inhibitory phenomenon along with the increase that MPB measures, therefore (C schemes to grope suitable MPB consumption GSV consumption to be fixed on 25ng and 50ng, D schemes).
Fig. 6: the causal result showing DOX checking high transfer pancreas cancer cell strain M45 transfer characteristics and transgenation.A, C, for after inoculation M45, dissect the lungs under the naked eyes of per os feeding DOX nude mice and liver, and B, D do not add nude mice lungs and liver under the naked eyes of DOX group in application water.
Fig. 7: the causal result showing DOX checking high transfer pancreas cancer cell strain M45 transfer characteristics and transgenation.A, C are for after inoculation M45, dissect lungs and the liver of (20 ×) under the microscope of per os feeding DOX nude mice, B, D are (20 ×) nude mice lungs and livers under not adding the microscope of DOX group in application water, and in figure, arrow is depicted as metastasis.
Fig. 8: the result showing gene clone and location.Be the GSV two ends PCR primer size by obtaining after splinkette PCR in A, B carries out the assignment of genes gene mapping after PCR primer being checked order.
Embodiment
The object of the invention is to the defect overcoming prior art, invent the method for a kind of random gene sudden change regulation and control and use the method to screen the gene of the transfer controlling carcinoma of the pancreas and/or other malignant tumours.The feature of the method comprises: can produce the sudden change of full-length genome homozygous gene; Full-length genome genescreen and gene function analysis are provided; Find simultaneously and confirm the relation of its transgenation and functional expression; Systemic gene functional localization and analyze its heredity and biochemical pathway in functional characteristics; Sharp separation and disease related gene and rheostatic type gene expression regulation.
The party's ratio juris is: by based on the piggyBac transposon (GI:226433913) of high effective integration building gene and search carrier (gene search vector in eukaryotic cell, GSV), by its radom insertion genome, tsiklomitsin response element (TRE wherein entrained by GSV, GI:76667907) two sections of tsiklomitsin response element splicings are preferably utilized to obtain, the activation of such activating transcription factor to it is stronger, start the distance of transcribing farther, the activation of activating transcription factor tTA can be subject to, piggbac transposon is energy high effective integration in eukaryotic cell, and integration site is partial to encoding gene, and seamless excision can be carried out by transposase at insertion postgenome, these all meet the requirement that a desirable gene searches carrier.Under the effect of tTA, the 14Tet in GSV can start transcribing of neighbouring DNA sequence dna very by force; If the RNA transcribed out is contrary with upstream gene transcriptional orientation, then can obtain sense-rna, be combined with the mRNA of upstream gene thus stop the accurate translation of this gene; If direction is identical, then can process LAN downstream gene, therefore can by the transgenation near insertion point by this carrier.Can also comprise neomycin resistance gene in GSV, this resistant gene can utilize G418 screening positive clone in eukaryotic cell, shows as kalamycin resistance at prokaryotic cell prokaryocyte, for screening positive bacterium colony.Certainly, the resistant gene in described GSV can be suitable for the original paper that can carry out resistance screening in eukaryotic cell and prokaryotic cell prokaryocyte respectively for any.In addition, the combination of activating transcription factor tTA and TRE is also subject to the regulation and control of tetracycline medication such as doxycycline (Doxcyclin), whether activate TRE and start by adding or do not add regulation and control tTA that DOX can be artificial and transcribe, thus can find simultaneously and confirm the relation of its transgenation and functional expression, therefore be a kind of sudden change control technique, its principle is as shown in Figure 1.Those skilled in the art know, in order to realize or realize object of the present invention better, gene of the present invention searches carrier can also comprise Plasmid replication origins, and this Plasmid replication origins there is no particular requirement, as long as it can effective starting plasmids copying in corresponding host, as p15A replicon; Be beneficial to the multiple clone site that exogenous sequences inserts; The tsiklomitsin response element of goal of regulation and control genetic transcription and translation, as TRE; Can in eukaryotic cell the transposon of random integration, as PiggyBac; Be beneficial to the ampicillin resistance gene screening positive bacterium colony in prokaryotic system; Energy effectively start gene searches the promotor that carrier is translated at host cell transcription, as P-CMV; And other plasmid common element, as enhanser, fluorescence protein gene, the terminator of being convenient to genetic expression detection, etc.
The present invention carries out by following technical solution: the gene set up based on piggyBac transposon searches carrier; Set up human pancreas cancer Aspc-1 cell strain Tet-off expression regulation system and High-efficiency gene search vector method; Build Aspc-1 cell full-length genome homozygous gene mutant cell storehouse; High transfer pancreas cancer cell strain after screening sudden change in nude mouse; Primary culture in vitro metastasis; Verify the cause-effect relationship (specific transcriptional incitant activity activator or inactivation by Tetracycline regulation) of high-transfer cell strain and the transgenation filtered out; Cloning and identification candidate gene, is shown in Fig. 2.
As well known to those skilled in the art, ensureing under the precursor realizing the object that gene is searched, described gene searches carrier can also comprise other functional element, described functional element is all effectively connected, to realize being replicated in prokaryotic host cell, increasing, detecting, screening, isolated or purified, and/or be replicated in eukaryotic host cell, increase, integrate with host genome, transcribe, translate, detect, screen, the object of isolated or purified.Simultaneously, as well known to those skilled in the art, ensureing that, under the prerequisite realizing the object that gene is searched, the order that described gene searches the element of carrier can suitably adjust, such as, the position of described protokaryon resistant gene and eucaryon resistant gene can be exchanged mutually.Such distortion is also included within scope of the present invention.
It will be appreciated by those skilled in the art that gene of the present invention searches in carrier each element used is all functional elements well known in the art, and its concrete nucleotide sequence can find in database known in the art easily and be suitable for.
As well known to those skilled in the art, the method of the gene that random gene regulate and control method of the present invention, the method for screening height transfer and/or low transfer human tumor cell line, screening raising and/or reduction human tumor cell line shift is not only applicable to human pancreatic carcinoma cells system, is applicable to other people tumor cell line yet.
The full-length genome libraries of random mutants that the method for the invention obtains produces at random and mutant character can be hereditary, the gene of the sudden change comprised covers whole genome from probability, the to be detected controlling gene acting on objective trait of screening or signal path can be applied to, described objective trait, such as but not limited to acceleration Nasopharyngeal neoplasms, reduction Nasopharyngeal neoplasms and the generation of screening tumour, resistance etc., also screens the cell of certain specific molecular marker of cell surface expression by flow cytometry FACS.
As well known to those skilled in the art, the gene that the method for the invention obtains can be applied to the preparation of antitumor drug, and now, described gene is the gene of inhibition tumor cell transfer.Described gene can be prepared according to the preparation form of known gene therapy medicament, such as described gene can connect into adenovirus carrier to be prepared with the form of recombinant vectors, or described gene can connect into protokaryon or carrier for expression of eukaryon, in corresponding host cell, expression, purifying, provide with the form of the protein of purifying.
As well known to those skilled in the art, the inhibitor of the gene that the method for the invention obtains can be applied to the preparation of antitumor drug, and now, described gene is the gene promoting Nasopharyngeal neoplasms.The inhibitor of described gene can be the siRNA utilizing RNA interference principle to realize therapeutic purpose.For design and the preparation method of such siRNA, this area has had a great selection of document can be for reference.The inhibitor of described gene also can be the functional element of transcribing and/or expressing that microRNA etc. directly suppresses target gene.
As well known to those skilled in the art, the inhibitor of the protein expression product of the gene that the method for the invention obtains can be applied to the preparation of antitumor drug, and now, described gene is the gene promoting Nasopharyngeal neoplasms.The inhibitor of the protein expression product of described gene can be the antibody of the protein expression product neutralizing described gene.Described antibody can be monoclonal antibody, also can be polyclonal antibody.
As well known to those skilled in the art, the tumour that the method for the invention can be treated never is only limitted to the carcinoma of the pancreas of the present invention following embodiment institute illustration.Because principle of the present invention is the controlling gene that the surveyed performance screening of the metastases proterties utilizing tumour cell to occur after transfection carrier of the present invention is corresponding with it, therefore measured gene is directly functionally relevant to the tumor cell line of used detection.Because carrier of the present invention there is no particular requirement to used tumor cell line, therefore, the tumour that medicine of the present invention can be treated also there is no special restriction.
To further illustrate the present invention by following non-limiting example below, as well known to those skilled in the art, without departing from the spirit of the invention, can make many amendments to the present invention, such amendment also falls into scope of the present invention.
Following experimental technique if no special instructions, is ordinary method, and the experiment material used if no special instructions, all can easily obtain from commercial company.
Embodiment
Embodiment 1 major experimental material
Restriction enzyme, T4DNA ligase enzyme is purchased from NEB company, primer, order-checking, Taq polysaccharase is all purchased from Invitrogen company, plasmid extraction kit, gel reclaims test kit purchased from OMEGA company, Aspc-1 cell strain (purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell centre), foetal calf serum, DMEM substratum, PBS is purchased from Hyclone company, FugenHD is purchased from Roche company, various cell cultures consumptive material is all purchased from Corning company, tetracycline (puromycin), Luciferin substrate is purchased from Invivo company, Liu Suanyan NEOMYCIN SULPHATE (neomyicn, G418) purchased from Merck company, thymus gland defect nude mice (BALB/c nu/nu) female mouse is purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
Embodiment 2: gene searches the establishment of carrier
In the present invention, gene search carrier comprises several parts such as tsiklomitsin response element, neomycin resistance gene, Plasmid replication origins and piggbac transposon, and need these cores to be stitched together, main-process stream is shown in Fig. 3 A, specifically comprises following components:
The acquisition of 1.TATA-14Tet fragment, this part comprises eukaryotic transcription starting point TATA box and 2 tsiklomitsin response elements; Utilize Restriction enzyme Sma I, XhoI and EcoRI singly to cut respectively and twoly cut pUHD13-3 plasmid
38, method is as follows:
SmaI is mono-cuts plasmid pUHD13-3, reaction system as described in Table 1:
Table 1:
Reaction conditions is: hatch 2h for 25 DEG C.
XhoI and EcoRI is two cuts plasmid pUHD13-3, reaction system as described in Table 2:
Table 2:
Reaction conditions is: 37 DEG C of water-bath 2h.
Gel reclaims purifying respectively and is about 3kb and 0.45kb digestion products, by reclaim after product 16 DEG C of connections spend the night, reaction system as described in Table 3:
Table 3:
Reaction conditions is: 16 DEG C of connections are spent the night.
Ethanol precipitation purifying is utilized to connect product, the product precipitation DDH of purifying
2o dissolves.
The conventional electric shocking method of connection product utilization after purifying is converted into DH10B bacterium (invitrogen), utilizes the 10cm agar plate screening positive clone with ammonia benzyl resistance.
OMEGA company plasmid extraction kit is utilized to extract plasmid after resistant clones amplification cultivation on picking flat board, plasmid after extracting is carried out the reaction of XhoI+XbaI double digestion, and purifying reclaims about 0.8kb product, obtains TATA-14Tet fragment, concrete grammar is the same, to see in Fig. 3 A 1. part.
2.Neo-PA-Ori fragment obtains, and utilizes NheI+XbaI double digestion pACYC184 plasmid
39, purifying reclaims the product Ori of about 0.8kb; Utilize primer Neo-F and Neo-R to carry out pcr amplification Neo-PA fragment, reaction system is as shown in following table 4 and table 5:
Table 4:
Table 5:
PCR primer is carried out NheI is mono-to be cut, reclaim after purifying be connected with Ori fragment, transform, with kantlex carry out colony clone plate screening, choose clone, plasmid extraction and utilize XhoI+NheI double digestion, reclaim purifying after obtain the Neo-PA-Ori fragment that length is about 2.1kb, to see in Fig. 3 A 2. part.
3.TATA-14Tet the splicing of fragment and Neo-PA-Ori fragment, enzyme is cut above-mentioned two fragments after purifying recovery, connect, be converted into DH10B, utilize kantlex to screen, choose clone to cultivate, extract plasmid, gained plasmid BamHI enzyme is cut rear recovery, and method therefor is the same, obtain the fragment TATA-14Tet-Neo-PA-Ori being about 2.9kb, to see in Fig. 3 A 3. part.
4. TATA-14Tet-Neo-PA-Ori fragment is inserted piggyBac transposon
40in, to see in Fig. 3 A 4. part, utilize primer PB-P1 and PB-P2 to carry out pcr amplification piggyBac empty carrier
40part, BamHI enzyme is cut, reclaim after to be connected with fragment and transform DH10B, choose clone, upgrading grain, BamHI enzyme cut and identify that correct clone is inserted in direction, namely enzyme obtains after cutting being about 6kb+0.8kb fragment, but not 2.1kb+4.7kb fragment, and concrete grammar is the same.
5. CMV promoter is inserted before Neo, utilize primer Np5 and Np3 from PBR-β-Neo
39on amplify CMV promoter fragment, be about 0.3kb, two the cutting of BamHI+BglII is connected with the fragment obtained through the mono-PB-TATA-14Tet-Neo-PA-Ori of the cutting plasmid of BamHI afterwards, concrete grammar is the same, the gene finally obtaining being about 7.1kb after qualification searches carrier (GSV), to see in Fig. 3 A 5. part, plasmid map is shown in Fig. 3 B.
Embodiment 3: the structure of full-length genome libraries of random mutants
Structure libraries of random mutants involved in the present invention is that the core parts searched on carrier by the gene based on piggyBac transposon of radom insertion cellular genome are played a role, this work comprise the carrier of construction expression activating transcription factor tTA, the pancreas cancer cell strain setting up Tet-off expression regulation system, random integration gene search carrier enter in Tet-off cell strain, specific as follows:
1. the plasmid of construction expression activating transcription factor tTA, in order to set up the Tet-off cell strain of high expression tTA, first inserts strong promoter CAG by tTA encoding gene front.Utilize primer tTA-P1 and primer tTA-P2 to amplify the encoding sequence of activating transcription factor tTA from pTet-off/hygro plasmid (purchased from Clontech), be connected into after BamHI and NotI double digestion equally through the expression vector pCAG-PBase of double digestion
41obtain expression vector CAG-tTA after conversion, Screening and Identification, order-checking, concrete grammar is the same.After being extracted in a large number by the CAG-tTA plasmid obtained, utilize KpnI enzyme to be cut into linearly, utilize sepharose reclaim test kit reclaim after, measure concentration, be stored to-20 DEG C for subsequent use.
2. set up the Aspc-1 cell strain of the Tet-off of stably express.
2.1 cell transfectings: by linear CAG-tTA plasmid transfection Aspc-1 cell, utilize the Tet-off cell strain of luciferase reporter gene technology screening high expression.According to Roche FugeneHD specification sheets, in day before transfection inoculation 5 × 10
5aspc-1 cell is to 60mm Tissue Culture Dish, when cell grows to about 80%, with the DMEM substratum of serum-free antibiotic-free, CAG-tTA plasmid is diluted to 0.02 μ g/ μ l, get 250 μ l, add 15 μ l FugeneHD wherein, mix gently, after normal temperature hatches 15min, slowly instill in off-the-shelf cell, mix gently, place the CO of 37 DEG C
2continue in incubator to cultivate; Peptic cell after 24h, in average mark to a 3 10cm culture dish, adds in the substratum of purine-containing mycin (puromycin, 2 μ g/ml) and continues to cultivate, change a not good liquor continue screening every 2 days; The clone that naked eyes can be observed is formed after 2 weeks.
The Aspc-1 cell strain of Tet-off of 2.2 luciferase reporter genes screening stably express, by the clone's picking after tetracycline screening and enlarged culturing, digests and inoculates 10
4cell is in 96 orifice plates, each clone's inoculation 6 parallel holes, overnight incubation in the 37 DEG C of incubators being placed in 5%CO2, saturated humidity, when cell density reaches 80%, by FuGeneHD specification sheets by aforementioned same procedure to transfection Luciferase reporter gene-pUHC13-3 plasmid in cell
38, every hole adds plasmid DNA 0.05 μ g, and FuGeneHD: DNA, by 3: 1 transfections, then adds doxycycline (Doxycycline, DOX) solution (0.2 μ g/ hole) in a half bore of each clone; Be placed in incubator to cultivate, with ice-cold PBS washed cell after 48 hours, supernatant discarded, utilize HarvestBuffer lysing cell (4 DEG C, 30min), after being mixed by lysate, every hole is got in 50 μ l to 96 hole enzyme plates, according to the proportions appropriate reaction liquid of ATP: Luciferin Buffer=1: 3.6, then on bioluminescent detection instrument, read absorbance, added each Kong Yuwei of DOX according to result calculating and added the downward multiple that luciferase expression intensity is compared in each hole of DOX; In selected clone, the fluorescence intensity of clone 2B6,2D2,5A3 is relatively high, and after adding DOX, its relative light unit can significantly decline, and the difference added before and after DOX can reach hundreds of to several thousand times, sees Fig. 4.
3. build full-length genome libraries of random mutants, utilize lipofection cotransfection gene to search carrier GSV and transposase MPB plasmid
37, under the help of mpb, GSV energy high effective integration is on genome; More satisfactory library should be the integration of each mutant clon only containing a GSV in library, in order to reduce the copy number be inserted in cell, needs the strict amount controlling the GSV of transfection; In addition, in order to make GSV high effective integration, ratio and the amount of GSV and MPB are also particularly important, and the MPB having bibliographical information excessive can greatly reduce the integration efficiency of GSV on the contrary; Therefore, before structure libraries of random mutants, need to grope the suitable amount of GSV and MPB.Preceding method is utilized to inoculate 10
5cell to 24 orifice plate, experiment sets up blank group; Independent transfection GSV group, is subdivided into 0ng, 25ng, 50ng, 100ng, 150ng, 200ng by different GSV amount; GSV and MPB cotransfection group, comprise fix respectively GSV be 25ng and 50ng, MPB amount change from 0ng, 25ng, 50ng, 100ng, 150ng, 200ng; Final DNA total amount is 500ng, uses carrier DNA polishing; Cell is proceeded in 10cm culture dish after transfection 24h, add the Liu Suanyan NEOMYCIN SULPHATE (neomycin of 400 μ g/ml, G418) screen, every 2-3 days changes a not good liquor, 3 Zhou Houke see blank group complete cell death, independent transfection 25ngGSV group colony counts is less, about 100, cotransfection 25ngMPB rear clone number increases substantially, up to about 1500, namely MPB significantly can improve the integration efficiency of GSV, obviously can find out difference, see Fig. 5 A after the clone after the medicine that sieves is utilized methylene blue staining.
The colony counts statistics obtained after concrete single transfection GSV group and cotransfection GSV and MPB group is shown in Fig. 5 B, 5C and 5D, can therefrom find out, the number of clone is closely related with the amount of GSV, and also there is extra-inhibitory phenomenon to GSV in MPB, when MPB: GSV more than 1: 1, the colony counts obtained reduces rapidly.Therefore, in order to reduce the copy number of GSV in individual cells, taking into account the total clone's number after cotransfection MPB simultaneously, GSV and MPB being fixed on 50ng and carrying out library construction.According to aforementioned proportion, every 10
5the each 50ng of cotransfection GSV and mPB in cell, after transfection every 10
5cell proceeds in a 10cm culture dish and adds Liu Suanyan NEOMYCIN SULPHATE screening about 3 weeks, carries out altogether 8 experiments, obtains the clone that about 1,200,000 have G418 resistance altogether, namely obtain comprising the mutated library that about 1,200,000 incorporate the clone of GSV.
Embodiment 4: screening in the body of high transitivity pancreas cancer cell strain
The mutant clon number that aforementioned constructed mutated library comprises is more than 40 times of whole cellular genome encoding gene number, therefore the gene suddenlyd change covers whole genome from probability; This library is divided into four groups, is L123, L456, L7 and L8 respectively, and often group is approximately containing 300,000 mutant clons; The BALB/C nude mice in all for 4-6 ages is also divided into four groups, often organize corresponding one group of mutated library, nude mice often organizes 9, and 3 for tail vein injection (10 simultaneously
6/ only), 3 are carried out intraperitoneal inoculation (3 × 10
6/ only), remainder 3 is for subcutaneous injection (4 × 10
6/ only); Peptic cell, utilizes PBS to wash 3 times, removes serum to reduce immunological rejection as far as possible, is placed on ice by cell, and counting cells also utilizes trypan blue to calculate cell survival rate, cell survival rate > 90% before guarantee inoculation; Cancer cells after utilizing 1ml syringe to inoculate sudden change, removes syringe needle to reduce its physical abuse effect to cell, air during tail vein injection in emptying syringe when drawing cell; The life state of close observation nude mice after inoculation, during ill sign to appear, puts to death mouse and carries out original cuiture.In about 1-3 month, mouse all shows exception, and after disconnected neck is put to death, iodine disinfection mouse is also dissected, each internal organs of visual inspection, cut metastatic tumor after seeing metastasis, utilize scalpel to shred, be suspended in centrifuge tube with substratum, the centrifugal 5min of 1000rpm, abandon supernatant, add 10ml erythrocyte cracked liquid, suspension cell lysing cell 10min, centrifugal, PBS wash 2 times, substratum resuspended after go in 10cm culture dish, place 37 DEG C of 5%CO
2cultivate in incubator; Second day replaced medium, remove not adherent suspended tissue, screening of medicaments (G418:400 μ g/ml is added after about one week, puromycin:2 μ g/ml), every 3-5 days changes liquid once to remove mesenchymal cell, vascular endothelial cell etc., next step checking work is carried out after cytotostatic growth is gone down to posterity, screening obtains 14 high transfer pancreas cancer cell strains altogether, the standard of high transfer is compared with the control, after the tumor cell inoculation of same quantity, contrast can not form transfer, and screening obtain can form metastasis in same time, see following table 6.
Table 6
Embodiment 5: the causal checking of transgenation and cancer metastasis
One of advantage of the present invention directly to verify the function of gene, carrys out the effect that blocking gene searches carrier, thus make to be reopened by the gene suddenlyd change or close by adding DOX; The Aspc-1 cell that screening obtains having high transfer proterties is directly verified by Inoculation.Get the BALB/C nude mice in 20 4-6 all ages, be divided into four groups, often organize 5, enlarged culturing high-transfer cell strain M45, peptic cell, utilizes PBS to wash 3 times, for reducing the rejection of mouse, removes serum, counting cells as far as possible, by 2 × 10
5/ only carry out mouse tail vein injection 10, wherein 5 mouths feed doxycycline DOX (200 μ g/ml), do not feed, change a not good liquor every three days for another five, simultaneously by 2 × 10
5/ only carry out mouse spleen to inject 10, first Amobarbital (50mg/kg) intraperitoneal injection of anesthesia mouse is utilized, then osculum is cut off in belly left side central portion, tear skin, expose stomach wall, red color visible strip spleen, mentions spleen gently with tweezers, along spleen y direction inserting needle, slowly push cell suspension 100 μ l (2 × 10
6/ ml), extract syringe needle after having injected, spleen is pushed back intraperitoneal gently, peritoneal suture and skin, until mouse revives under mouse is placed on warm environment, puts back in cage and continue to raise, observe mouse, when ill sign appears in mouse, put to death, dissect mouse, each Organ relative weight situation of visual inspection mouse, tail vein injection main detection lung tumors shifts, and the transfer case of tumour cell in liver is observed in spleen inoculation, after Taking Pictures recording, takes off each organ, is immersed in 4% formalin stationary liquid, carry out paraffin section-HE after to be fixed to dye, basis of microscopic observation shifts, get fixing tissue block, dewater with LEICAASP 200S automatic dehydrator, transparent, waxdip, afterwards with the embedding of LECIA EG1500H embedding machine, LECIA EG1500C cold bench cools, at LECIA RM2255 microtome, paster, roasting sheet, finally carry out HE dyeing with on LECIA Stainer XL automatic staining machine, observe after mounting, dehydration, transparent and waxdip program (LEICA ASP 200S) is as follows: 70% ethanol × 1h, 80% ethanol × 1h, 90% ethanol × 1h, 95% ethanol I × 45min, 95% ethanol II × 45min, dehydrated alcohol I × 40min, dehydrated alcohol II × 40min, dimethylbenzene I × 5min, dimethylbenzene II × 5min, wax cup I × 1h, wax cup II × 1h, wax cup III × 1h, HE dyeing procedure (LECIA Stainer XL) is as follows: dimethylbenzene I × 10min, dimethylbenzene II × 10min, dehydrated alcohol I × 1min, dehydrated alcohol II × 1min, 95% ethanol × 1min, 90% ethanol × 1min, 70% ethanol × 1min, brazilwood extract dyeing liquid × 1min, acidic alcohol × 30s, ammoniacal liquor × 30s, 0.5% eosin stains liquid × 20s, 80% ethanol × 1min, 90% ethanol × 25s, 95% ethanol × 30s, 95% ethanol × 30s, dehydrated alcohol I × 30s, dehydrated alcohol II × 1min, dimethylbenzene I × 2min, dimethylbenzene II × 2min, observe after neutral gum mounting.After dissecting, naked eyes are visible, and do not feed the mouse of DOX visible metastasis on lung and on liver, and add DOX in tap water after, the metastasis of lungs and liver is not obvious, naked eyes, HE coloration result of cutting into slices are shown in Fig. 6 and Fig. 7.
Embodiment 6: the molecular cloning of candidate gene
Screen high transitivity pancreas cancer cell strain and after carrying out functional verification, next step clone gene searches the gene of carrier sudden change.The sequence of GSV is known, method by Splinkette PCR clones the sequence around GSV, again by blast comparison (http://genome.ucsc.edu/cgi-bin/hgBlat? command=start) find the gene of this position, to carry out in Splinkette PCR experiment the primer as described in Table 7:
Table 7:
Extract according to genome DNA extracting reagent kit (prestige lattice Lars, DNAVzol) specification sheets the cell strain genomic dna screened, utilize Sau3AI enzyme to cut 1 μ g genomic dna, specifically reaction is as described in Table 8:
Table 8:
Reaction conditions is: 37 DEG C of enzymes cut through night; Be connected with joint (Adaptor) after 65 DEG C of inactivation 20min, respectively get 5 μ l SPL and SPR-Sau3 AI (100 μm), add 90 μ l water, after being heated to 100 DEG C of reaction 10min, slowly cooling to the product after normal temperature is joint, frozen to-20 DEG C for subsequent use, ligation is specific as follows to be stated shown in table 9:
Table 9:
Reaction conditions is: 16 DEG C of connections are spent the night; Carry out first round PCR reaction, reaction system as described in Table 10:
Table 10:
Reaction conditions is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 45s, 68 DEG C of annealing 2min, react 30 circulations; 94 DEG C of sex change 20s, 60 DEG C of annealing 30s, 72 DEG C extend 2min; React 34 circulations; 72 DEG C extend 5min.Carry out second step PCR reaction after reaction terminates, reaction system is as shown in following table 11:
Table 11:
Reaction conditions is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 20s, 61 DEG C of annealing 30s, 72 DEG C extend 1min, react 40 circulations; 72 DEG C extend 5min.After reaction terminates, sepharose reclaims PCR primer and checks order; After obtaining 5 ' end and 3 ' end DNA sequence dna, log in UCSC genome browser and carry out Blat comparison, the position of GSV on genome, location, analyzes it and causes the direction of transcribing, for M45, see Fig. 8; The gene totally 19 of clone, sees the above table.
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Claims (1)
1. screen a method for high-transfer human tumor cell line, it comprises step:
1) with cag-tTA plasmid transfection human tumor cell line, the Tet-off cell strain of stably express activating transcription factor is obtained;
2) gene is searched the plasmid of carrier and effective expression transposase MPB, according to each 50ng/10
5the transfection amount of individual cell, cotransfection step 1) the Tet-off cell strain that obtains, obtain full-length genome cell homozygous mutant;
3) by the step 2 of significant quantity) the middle full-length genome cell homozygous mutant inoculation BALB/C nude mice obtained;
4) put to death BALB/C nude mice after growing tumour in BALB/C nude mouse to be seeded or manifesting morbid state, be separated and obtain metastasis and carry out primary culture in vitro, obtain the human tumor cell line of high transfer and/or low transfer compared with the human tumor cell line contrasted;
5) function of gene is verified, carry out by adding DOX the effect that blocking gene searches carrier, thus make to be reopened by the gene suddenlyd change or close; The Aspc-1 cell that screening obtains having high transfer proterties is directly verified by Inoculation;
Wherein said gene is searched carrier and is comprised tsiklomitsin response element, piggybac transposon, neomycin resistance gene and Plasmid replication origins, and described tsiklomitsin response element is obtained by two sections of tsiklomitsin response element splicings,
The plasmid of wherein said effective expression transposase MPB is mpb,
Described human tumor cell line is human pancreas cancer Aspc-1 cell strain.
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