CN111850037A - High-throughput genetic screening method for piggyBac transposon - Google Patents
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Abstract
The invention provides a piggyBac transposon high-throughput genetic screening method, which comprises the following steps: 1) constructing a PB [ tet-on-SD ] vector, which comprises piggyBac defective transposon, trans-tetracycline regulation and control activator rtTA expression frame and a splicing donor regulated and started by a tetO operon; 2) the PB [ tet-on-SD ] plasmid and the plasmid for coding piggyBac transposase are transfected into in vitro culture cells simultaneously; 3) adding a tetracycline derivative doxycycline medicament into a culture medium to induce the over-expression of an endogenous gene; 4) the cell phenotype is analyzed, and the insertion mutation positioning is carried out on the cell clone with the specific phenotype, so as to determine the gene which can cause the specific phenotype after the overexpression. The high-throughput genetic screening method of the piggyBac transposon can efficiently and conveniently carry out high-throughput genetic screening of whole genome overexpression in vitro culture cells.
Description
Technical Field
The invention relates to a piggyBac transposon high-throughput genetic screening method, belonging to the technical field of biology.
Background
High throughput genetic screening is the discovery of genes that function in a particular biological process by efficiently mutating the genome of individuals or cells on a large scale and then screening the mutated individuals or cells for a particular biological phenotype. High throughput genetic screening is widely used in functional genome research, drug target exploration and drug resistance mechanism research.
Gene mutations can result in loss-of-function or gain-of-function of a gene. Gene overexpression is one of the gain-type gene mutations. Currently, high-throughput gene overexpression genetic screening mainly employs a method of introducing a cDNA library into cells using lentivirus. The method relates to the steps of cDNA library establishment, lentivirus packaging and the like, and is relatively complex to operate. The tissue origin and quality of cDNA libraries directly limit the genomic coverage of genetic screens.
To date, there is a lack of a method for efficiently and conveniently performing high-throughput genetic screening for whole genome overexpression in vitro cultured cells in the field of biology.
Disclosure of Invention
The invention aims to provide a method for randomly inducing endogenous gene overexpression to carry out high-throughput genetic screening based on piggyBac transposon. So as to efficiently and conveniently carry out overexpression high-throughput genetic screening in vitro cultured cells.
The invention adopts the following technical scheme:
a piggyBac transposon high-throughput genetic screening method is characterized by comprising the following steps:
1) constructing a PB [ tet-on-SD ] vector, which comprises piggyBac defective transposon, trans-tetracycline regulation and control activator rtTA expression frame and a splicing donor regulated and started by a tetO operon;
2) The PB [ tet-on-SD ] plasmid and the plasmid for coding piggyBac transposase are transfected into the in vitro culture cell simultaneously, and the PB [ tet-on-SD ] vector is mediated to be randomly inserted into the genome of the in vitro culture cell;
3) adding a tetracycline derivative doxycycline medicament into a culture medium to induce the over-expression of an endogenous gene;
4) the cell phenotype is analyzed, and the insertion mutation positioning is carried out on the cell clone with the specific phenotype, so as to determine the gene which can cause the specific phenotype after the overexpression.
Further, in the piggyBac transposon high-throughput genetic screening method, elements contained in the PB [ tet-on-SD ] vector are as follows from the 5 'end to the 3' end:
PBR: piggyBac transposon right arm;
UBC: the human ubiquitin C promoter;
rtTA: trans-tetracycline-regulated activators;
IRES: an internal ribosome entry site sequence;
puro: puromycin resistance gene;
WPRE: drought hepatitis virus post-transcriptional regulatory elements;
PA: rabbit β -globin polyA;
ins: insulator sequence;
tetO: a tetracycline operator sequence;
SD: a splice donor sequence;
PBL: left arm of piggyBac transposon.
Further, the piggyBac transposon high-throughput genetic screening method of the invention also has the following characteristics: in step 4), the step of analyzing the phenotype of the cells comprises the step of performing genetic screening.
Further, the piggyBac transposon high-throughput genetic screening method of the invention also has the following characteristics: wherein, in the step 2), the in vitro cell adopts genetic screening to obtain PLX4032 drug-resistant BRAF-carrying cellsV600KMutated melanoma cell clones.
Further, the piggyBac transposon high-throughput genetic screening method of the invention also has the following characteristics:
in the step 1), the construction of the PB [ tet-on-SD ] vector comprises the following steps:
step 1-1, synthesizing LUN-SD sequence, and inserting enzyme digestion into the positions of AgeI and BglII of defective piggyBac transposon to obtain PB [ SD ] vector.
Further, the piggyBac transposon high-throughput genetic screening method of the invention also has the following characteristics:
in the step 1), the construction of the PB [ tet-on-SD ] vector comprises the following steps:
step 1-2, synthesizing UBC-rtTA-IRES-puro-WPRE sequence (SEQ ID No: 2), and performing enzyme digestion and insertion between MluI and PstI sites of the PB [ SD ] vector to obtain the PB [ rtTA, SD ] vector.
Further, the piggyBac transposon high-throughput genetic screening method of the invention also has the following characteristics:
in the step 1), the construction of the PB [ tet-on-SD ] vector comprises the following steps:
and (1) amplifying a rabbit beta-globin polyA sequence by using primers PAF and PAR and taking a pCAG-EGFP plasmid as a template, and carrying out enzyme digestion on PstI to insert a PstI site of a PB [ rtTA, tetO-SD ] vector to obtain a PB [ rtTA-PA, tetO-SD ] vector.
Further, the piggyBac transposon high-throughput genetic screening method of the invention also has the following characteristics: in the step 1), the construction of the PB [ tet-on-SD ] vector comprises the following steps:
step 1-4, synthesizing an Ins sequence (SEQ ID No: 7) NotI enzyme digestion filling-up, inserting a PB [ rtTA-PA, tetO-SD ] vector which is subjected to XbaI enzyme digestion filling-up, and finally obtaining a PB [ tet-on-SD ] vector.
Further, the piggyBac transposon high-throughput genetic screening method of the invention also has the following characteristics:
in step 4), the step of positioning the insertion mutation of the cell clone with the specific phenotype is as follows:
the Splinkerette linker-ligated genomic DNA library was subjected to a first round of PCR amplification using piggyBac transposon arm specific primers (left arm: PBLlink1, SEQ ID No: 10) and linker specific primers (LinkAmp1, SEQ ID No: 11). The product obtained after amplification is subjected to a second round of nested PCR amplification by a piggyBac transposome arm specific primer (left arm: PBLlink2, SEQ ID No: 12) and a joint specific primer (LinkAmp2, SEQ ID No: 13). Nested PCR amplification products were used for Sanger sequencing. And comparing the genome sequence around the insertion site obtained by sequencing with the sequence in the public genome database so as to determine the insertion site of the transposon.
Advantageous effects of the invention
The high-throughput genetic screening method of the piggyBac transposon can efficiently and conveniently carry out high-throughput genetic screening of whole genome overexpression in vitro culture cells.
Drawings
FIG. 1 is a schematic diagram of the PB [ tet-on-SD ] vector of the present invention.
FIG. 2 is a schematic diagram of a scheme for the in vitro culture of overexpression of endogenous genes in cells mediated by the PB [ tet-on-SD ] vector.
FIG. 3 is an example of the application of the high-throughput genetic screening for piggyBac transposon gene overexpression in the study of drug resistance mechanism.
Detailed Description
The following describes embodiments of the present invention with reference to the drawings.
In one embodiment of the invention, the whole genome overexpression high-throughput genetic screening is efficiently and conveniently carried out in vitro cultured cells, the piggyBac transposon carrying the tet-on system is used for inducing the conditional overexpression of endogenous genes beside a transposon insertion site, so that the genetic screening of drug resistance mechanism research is carried out.
In the preparation of the piggyBac transposon vector carrying the tet-on system of this example, the involved plasmid extraction, plasmid transformation, escherichia coli culture, PCR, enzyme digestion, Klenow enzyme filling-in, ligation are experiments well known to those skilled in the art. The general experimental conditions can be referred to in the fourth edition of the molecular cloning instructions, compiled by m.r. green and j.
The specific establishment steps of the piggyBac transposon vector PB [ tet-on-SD ] carrying the tet-on system are as follows:
synthesizing LUN-SD sequence shown in SEQ ID No: 1, enzyme cutting and inserting between AgeI and BglII loci of defective piggyBac transposons to obtain a PB [ SD ] vector.
The UBC-rtTA-IRES-puro-WPRE sequence was synthesized as shown in SEQ ID No: 2, cutting enzyme and inserting it between MluI and PstI sites of PB [ SD ] vector to obtain PB [ rtTA, SD ] vector.
The tetO sequence was synthesized as shown in SEQ ID No: 3, cutting enzyme and inserting the enzyme between XbaI and AgeI sites to obtain a PB [ rtTA, tetO-SD ] vector.
Primer PAF was used, see SEQ ID No: 4, and PAR, see SEQ ID No: and 5, amplifying a rabbit beta-globin polyA sequence by using pCAG-EGFP plasmid (purchased from Addgene) as a template, wherein the sequence is shown in SEQ ID No: 5, PstI is inserted into a PstI site of the PB [ rtTA, tetO-SD ] vector through enzyme digestion to obtain the PB [ rtTA-PA, tetO-SD ] vector.
The Ins sequence was synthesized as shown in SEQ ID No: 7, filling up by NotI enzyme, inserting the PB [ rtTA-PA, tetO-SD ] vector which is filled up by XbaI enzyme, and finally obtaining the PB [ tet-on-SD ] vector as shown in figure 1.
FIG. 1 is a schematic diagram of the construction of the PB [ tet-on-SD ] vector of the invention, the construction comprising the following elements in sequence from the 5 'end to the 3' end: PBR: piggyBac transposon right arm; UBC: the human ubiquitin C promoter; rtTA: trans-tetracycline-regulated activators; IRES: an internal ribosome entry site sequence; puro: puromycin resistance gene; WPRE: drought hepatitis virus post-transcriptional regulatory elements; PA: rabbit β -globin polyA; ins: insulator sequence; tetO: a tetracycline operator sequence; SD: a splice donor sequence; PBL: left arm of piggyBac transposon. After the PB [ tet-on-SD ] vector is introduced into cells, the PB [ tet-on-SD ] vector is randomly inserted into a cell genome, rtTA reading frame expresses rtTA protein, tetO is activated under Dox induction, and endogenous gene overexpression is mediated, as shown in figure 2.
In one example of high-throughput genetic screening of endogenous gene overexpression in vitro cultured cells by using a PB [ tet-on-SD ] vector, the PB [ tet-on-SD ] vector is used for drug resistance mechanism research of a melanoma targeting drug PLX 4032.
Carrier BRAF isolated from patientV600KThe mutated YUMAC melanoma cells are cultured on a culture dish in a monolayer adherent manner, and the components of a culture medium are as follows: OptiMEM (from Gibco), 5% calf serum (from Gibco), 1% double antibody. The incubator conditions were set at 37 ℃ and 5% CO290-95% humidity.
1ug each of the nucleic acid of PB [ tet-on-SD ] transposon and the nucleic acid encoding piggyBac transposase was mixed with 10. mu.L of LLIPOFECTAMINE 2000 liposomes (purchased from Invitrogen) in 500. mu.L of Opti-MEM medium. Subsequently, the cells were added dropwise to a 6-well plate containing 2mL of medium and cultured in a monolayer adherent manner. The following day cells from one well of a 6-well plate were transferred to a 10cm petri dish and 2. mu.g/ml puromycin (ex Invitrogen), 200ng/ml doxycycline (ex sigma), 1.5uM PLX4032 (ex sigma) was added to the medium and screened for two weeks to give transposon-carrying and PLX 4032-resistant YUMA cell clones.
In one example of the localization of insertional mutations to cell clones with a particular phenotype, Splinkerette-PCR was used to localize the position of the PB [ tet-on-SD ] transposon insertion in PLX 4032-resistant YUMAC cell clones.
Clones of PLX 4032-resistant YUMAC cells were cell expanded and genomic DNA was extracted using QIAGEN kit. After 2ug cell genome DNA is digested by Sau3AI enzyme in a 30 muL reaction system, Splinkerette joints are added at two ends of a DNA fragment at the same time, and the method comprises the following specific steps: SpLink1, as set forth in SEQ ID No: 8, and SpLink2, as shown in SEQ ID NO: 9, oligonucleotides were dissolved in NEB buffer No. 5X 2 to 50uM, 1: 1 SpLink1 and SpLink2 adaptor primer solutions were mixed to a final concentration of 25uM for each primer, and the mixture was frozen at-20 ℃. The adaptor primer mix was dissolved on ice and 1. mu.L of adaptor mix was used per 300ug of digested genomic product. On the PCR instrument, the adaptor primers were denatured first at 95 degrees for 5min, and then annealed to room temperature at a rate of 1 degree reduction every 15 seconds to form double-stranded adaptors. Finally, the linker was ligated to the Sau3AI digested genomic DNA fragment using T4 ligase in a 40. mu.L reaction.
The genomic DNA library linked to the Splinkerette linker was primed via piggyBac transposome arm specific primers, left arm: PBLlink1 as shown in SEQ ID No: 10, and a linker specific primer LinkAmp1, as shown in SEQ ID No: 11, a first round of PCR amplification was performed. And (3) carrying out amplification on the obtained product by using a piggyBac transposon arm specific primer, wherein the left arm: PBLlink2, as shown in SEQ ID No: 12, and a linker specific primer LinkAmp2, as shown in SEQ ID No: 13, a second round of nested PCR amplification was performed. Nested PCR amplification products were used for Sanger sequencing. And comparing the genome sequence around the insertion site obtained by sequencing with the sequence in the public genome database so as to determine the insertion site of the transposon.
As a result of this example, it was found that the transposon insertion mutation carried by one of the drug-resistant cell clones was localized to intron 10. Under the induction of Dox medicine, the result of fluorescent quantitative PCR (detection primers are SEQ ID No. 14 and SEQ ID No. 15, internal reference primers are shown as SEQ ID No. 16 and SEQ ID No. 17, and the C end of BRAF protein is over-expressed.
Fig. 3 shows the situation that BRAF protein is over-expressed by fluorescent quantitative PCR result under Dox drug induction. Genetic screening to obtain PLX4032 drug-resistant BRAF carried carrierV600KMutated melanoma cell clones, transposon insertional mutations were mapped to intron 10. Under the induction of Dox drugs, the result of fluorescent quantitative PCR shows that the C end of the BRAF protein is over-expressed.
In other embodiments of the invention, the PB [ tet-on-SD ] transposon system can be introduced into animal cells cultured in vitro by calcium phosphate transfection, polyethylene glycol-polyethyleneimine copolymer transfection, or electroporation transfection.
In other embodiments of the invention, the PB [ tet-on-SD ] transposon system can be used for functional genomic studies, drug target discovery and drug resistance mechanism studies.
Sequence listing
<110> Pan rain 22531
<120> high-throughput genetic screening method for piggyBac transposon
<130>JSP11901818
<160>17
<170>SIPOSequenceListing 1.0
<210>1
<211>191
<212>DNA
<213>artificial
<400>1
accggtcgct acttgcacca gaacgcccgc gaggatctct caggtaataa agagcgccaa 60
ggctggctgc aagcggagcc tctgagagcc tctgagggcc agggctactg cacccttggt 120
cctcaacgct ggggtcttca gaactagaat gctgggggtg gggtggggat tcggttccct 180
attccagatc t 191
<210>2
<211>3834
<212>DNA
<213>artifical
<400>2
acgcgtggcc tccgcgccgg gttttggcgc ctcccgcggg cgcccccctc ctcacggcga 60
gcgctgccac gtcagacgaa gggcgcagcg agcgtcctga tccttccgcc cggacgctca 120
ggacagcggc ccgctgctca taagactcgg ccttagaacc ccagtatcag cagaaggaca 180
ttttaggacg ggacttgggt gactctaggg cactggtttt ctttccagag agcggaacag 240
gcgaggaaaa gtagtccctt ctcggcgatt ctgcggaggg atctccgtgg ggcggtgaac 300
gccgatgatt atataaggac gcgccgggtg tggcacagct agttccgtcg cagccgggat 360
ttgggtcgcg gttcttgttt gtggatcgct gtgatcgtca cttggtgagt agcgggctgc 420
tgggctggcc ggggctttcg tggccgccgg gccgctcggt gggacggaag cgtgtggaga 480
gaccgccaag ggctgtagtc tgggtccgcg agcaaggttg ccctgaactg ggggttgggg 540
ggagcgcagc aaaatggcgg ctgttcccga gtcttgaatg gaagacgctt gtgaggcggg 600
ctgtgaggtc gttgaaacaa ggtggggggc atggtgggcg gcaagaaccc aaggtcttga 660
ggccttcgct aatgcgggaa agctcttatt cgggtgagat gggctggggc accatctggg 720
gaccctgacg tgaagtttgt cactgactgg agaactcggt ttgtcgtctg ttgcgggggc 780
ggcagttatg gcggtgccgt tgggcagtgc acccgtacct ttgggagcgc gcgccctcgt 840
cgtgtcgtga cgtcacccgt tctgttggct tataatgcag ggtggggcca cctgccggta 900
ggtgtgcggt aggcttttct ccgtcgcagg acgcagggtt cgggcctagg gtaggctctc 960
ctgaatcgac aggcgccgga cctctggtga ggggagggat aagtgaggcg tcagtttctt 1020
tggtcggttt tatgtaccta tcttcttaag tagctgaagc tccggttttg aactatgcgc 1080
tcggggttgg cgagtgtgtt ttgtgaagtt ttttaggcac cttttgaaat gtaatcattt 1140
gggtcaatat gtaattttca gtgttagact agtaaattgt ccgctaaatt ctggccgttt 1200
ttggcttttt tgttagacgc tagcggatcc ataacttcgt atagtataca ttatacgaag 1260
ttatgccacc atgtctaggc tggacaagag caaagtcata aacggagctc tggaattact 1320
caatggtgtc ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc tgggagttga 1380
gcagcctacc ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat 1440
cgagatgctg gacaggcatc atacccactt ctgccccctg gaaggcgagt catggcaaga 1500
ctttctgcgg aacaacgcca agtcataccg ctgtgctctc ctctcacatc gcgacggggc 1560
taaagtgcat ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg aaaatcagct 1620
cgcgttcctg tgtcagcaag gcttctccct ggagaacgca ctgtacgctc tgtccgccgt 1680
gggccacttt acactgggct gcgtattgga ggaacaggag catcaagtag caaaagagga 1740
aagagagaca cctaccaccg attctatgcc cccacttctg agacaagcaa ttgagctgtt 1800
cgaccggcag ggagccgaac ctgccttcct tttcggcctg gaactaatca tatgtggcct 1860
ggagaaacag ctaaagtgcg aaagcggcgg gccgaccgac gcccttgacg attttgactt 1920
agacatgctc ccagccgatg cccttgacga ttttgacctt gacatgctcc ccgggtaaat 1980
aacttcgtat agtatacatt atacgaagtt atggatccgc ggccgcaaat tccgcccctc 2040
tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt 2100
tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc 2160
tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca 2220
aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac 2280
gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg 2340
ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt 2400
gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct 2460
gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg 2520
ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt 2580
ggttttcctt tgaaaaacac gataatacca tggccaccga gtacaagccc acggtgcgcc 2640
tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg ttcgccgact 2700
accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc 2760
aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg 2820
gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg 2880
agatcggctc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg 2940
aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg 3000
tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg 3060
cggccgagcg cgctggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct 3120
tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca 3180
cctggtgcat gacccgcaag cccggtgcct gagttcgcgt ctggaacaat caacctctgg 3240
attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat 3300
gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt 3360
tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca 3420
ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg 3480
ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg 3540
aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca 3600
attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca 3660
cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc 3720
ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc 3780
agacgagtcg gatctccctt tgggccgcct ccccgcctgg aattaattct gcag 3834
<210>3
<211>290
<212>DNA
<213>artificial
<400>3
ctagacgagt ttactcccta tcagtgatag agaacgatgt cgagtttact ccctatcagt 60
gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta 120
ctccctatca gtgatagaga acgtatgtcg agtttatccc tatcagtgat agagaacgta 180
tgtcgagttt actccctatc agtgatagag aacgtatgtc gaggtaggcg tgtacggtgg 240
gaggcctata taagcagagc tcgtttagtg aaccgtcaga tcgcaccggt 290
<210>4
<211>27
<212>DNA
<213>artificial
<400>4
aaactgcaga attcactcct caggtgc 27
<210>5
<211>30
<212>DNA
<213>artificial
<400>5
ttaaaagctt gggctgcagg tcgagggatc 30
<210>6
<211>544
<212>DNA
<213>artificial
<400>6
gaattcactc ctcaggtgca gggctgccta tcagaaggtg gtggctggtg tggccaatgc 60
cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat 120
gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt 180
gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa 240
acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat 300
gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc 360
cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt 420
atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct 480
cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct 540
gcag 544
<210>7
<211>1229
<212>DNA
<213>artificial
<400>7
gcggccgcat ccccgggtac cgagttggcg cgcctggagc tcacggggac agcccccccc 60
caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc 120
ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca 180
gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt 240
tgagcctgca gacacctggg gggatacggg gaaaaagctt taggctgaaa gagagattta 300
gaatgacagg cgcgcctgga gctcacgggg acagcccccc cccaaagccc ccagggatgt 360
aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc 420
ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg 480
tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg 540
gggggatacg gggaaaaagc tttaggctga aagagagatt tagaatgaca ggcgcgccaa 600
ctcgaattac ggccggccgc atccccgggt accgagttgg cgcgcctgga gctcacgggg 660
acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc 720
agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag 780
cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt 840
ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaaagc tttaggctga 900
aagagagatt tagaatgaca ggcgcgcctg gagctcacgg ggacagcccc cccccaaagc 960
ccccagggat gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct 1020
ccgctccggt ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg 1080
gcacggggaa ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc 1140
tgcagacacc tggggggata cggggaaaaa gctttaggct gaaagagaga tttagaatga 1200
caggcgcgcc aactcgaatt agcggccgc 1229
<210>8
<211>61
<212>DNA
<213>artificial
<400>8
cgaagagtaa ccgttgctag gagagaccgt ggctgaatga gactggtgtc gacactagtg 60
g 61
<210>9
<211>48
<212>DNA
<213>aritificia
<400>9
gatcccacta gtgtcgacac cagtctctaa tttttttttt caaaaaaa 48
<210>10
<211>30
<212>DNA
<213>artificial
<400>10
cagtgacact taccgcattg acaagcacgc 30
<210>11
<211>28
<212>DNA
<213>artificial
<400>11
cgaagagtaa ccgttgctag gagagacc 28
<210>12
<211>30
<212>DNA
<213>artificial
<400>12
gagagagcaa tatttcaaga atgcatgcgt 30
<210>13
<211>25
<212>DNA
<213>aritficia
<400>13
gtggctgaat gagactggtg tcgac 25
<210>14
<211>21
<212>DNA
<213>artificial
<400>14
gctccagctt gtatcaccat c 21
<210>15
<211>20
<212>DNA
<213>artificial
<400>15
ggatgattga cttggcgtgt 20
<210>16
<211>17
<212>DNA
<213>artificial
<400>16
accgagcgcg gctacag 17
<210>17
<211>22
<212>DNA
<213>artificial
<400>17
cttaatgtca cgcacgattt cc 22
Claims (9)
1. A piggyBac transposon high-throughput genetic screening method is characterized by comprising the following steps:
1) constructing a PB [ tet-on-SD ] vector, which comprises piggyBac defective transposon, trans-tetracycline regulation and control activator rtTA expression frame and a splicing donor regulated and started by a tetO operon;
2) the PB [ tet-on-SD ] plasmid and the plasmid for coding piggyBac transposase are transfected into the in vitro culture cell simultaneously, and the PB [ tet-on-SD ] vector is mediated to be randomly inserted into the genome of the in vitro culture cell;
3) adding a tetracycline derivative doxycycline medicament into a culture medium to induce the over-expression of an endogenous gene;
4) the cell phenotype is analyzed, and the insertion mutation positioning is carried out on the cell clone with the specific phenotype, so as to determine the gene which can cause the specific phenotype after the overexpression.
2. The piggyBac transposon high throughput genetic screening method of claim 1, wherein:
wherein the elements contained in the PB [ tet-on-SD ] vector are as follows from the 5 'end to the 3' end:
PBR: piggyBac transposon right arm;
UBC: the human ubiquitin C promoter;
rtTA: trans-tetracycline-regulated activators;
IRES: an internal ribosome entry site sequence;
puro: puromycin resistance gene;
WPRE: drought hepatitis virus post-transcriptional regulatory elements;
PA: rabbit β -globin polyA;
ins: insulator sequence;
tetO: a tetracycline operator sequence;
SD: a splice donor sequence;
PBL: left arm of piggyBac transposon.
3. The piggyBac transposon high throughput genetic screening method of claim 1, wherein:
in step 4), the step of analyzing the phenotype of the cells comprises the step of performing genetic screening.
4. The piggyBac transposon high throughput genetic screening method of claim 1, wherein:
wherein, in the step 2), the in vitro cell adopts genetic screening to obtain PLX4032 drug-resistant BRAF-carrying cellsV600KMutated melanoma cell clones.
5. The piggyBac transposon high throughput genetic screening method of claim 1, wherein:
in the step 1), the construction of the PB [ tet-on-SD ] vector comprises the following steps:
step 1-1, synthesizing LUN-SD sequence, and inserting enzyme digestion into the positions of AgeI and BglII of defective piggyBac transposon to obtain PB [ SD ] vector.
6. The piggyBac transposon high throughput genetic screening method of claim 5, wherein:
in the step 1), the construction of the PB [ tet-on-SD ] vector comprises the following steps:
step 1-2, synthesizing UBC-rtTA-IRES-puro-WPRE sequence (SEQ ID No: 2), and performing enzyme digestion and insertion between MluI and PstI sites of the PB [ SD ] vector to obtain the PB [ rtTA, SD ] vector.
7. The piggyBac transposon high throughput genetic screening method of claim 6, wherein:
in the step 1), the construction of the PB [ tet-on-SD ] vector comprises the following steps:
and (1) amplifying a rabbit beta-globin polyA sequence by using primers PAF and PAR and taking a pCAG-EGFP plasmid as a template, and carrying out enzyme digestion on PstI to insert a PstI site of a PB [ rtTA, tetO-SD ] vector to obtain a PB [ rtTA-PA, tetO-SD ] vector.
8. The piggyBac transposon high throughput genetic screening method of claim 7, wherein:
in the step 1), the construction of the PB [ tet-on-SD ] vector comprises the following steps:
step 1-4, synthesizing an Ins sequence (SEQ ID No: 7) NotI enzyme digestion filling-up, inserting a PB [ rtTA-PA, tetO-SD ] vector which is subjected to XbaI enzyme digestion filling-up, and finally obtaining a PB [ tet-on-SD ] vector.
9. The piggyBac transposon high throughput genetic screening method of claim 1, wherein:
in step 4), the step of positioning the insertion mutation of the cell clone with the specific phenotype is as follows:
the Splinkerette linker-ligated genomic DNA library was subjected to a first round of PCR amplification using piggyBac transposon arm specific primers (left arm: PBLlink1, SEQ ID No: 10) and linker specific primers (LinkAmp1, SEQ ID No: 11). The product obtained after amplification is subjected to a second round of nested PCR amplification by a piggyBac transposome arm specific primer (left arm: PBLlink2, SEQ ID No: 12) and a joint specific primer (LinkAmp2, SEQ ID No: 13). Nested PCR amplification products were used for Sanger sequencing. And comparing the genome sequence around the insertion site obtained by sequencing with the sequence in the public genome database so as to determine the insertion site of the transposon.
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