CN109554447A - Integration site analysis method and primer of the slow virus carrier in CAR-T cell - Google Patents
Integration site analysis method and primer of the slow virus carrier in CAR-T cell Download PDFInfo
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Abstract
The integration site analysis method that the invention discloses a kind of slow virus carriers in CAR-T cell and applied to the primer sequence of this method.The method is to be unidirectional PCR using the area the LTR special primer of slow virus carrier to capture and be inserted into the area genome Shang LTR, then obtains integration site sequence as nested PCR amplification with two bell and spigot joint primers.Method of the invention is independent of restriction enzyme, and high sensitivity, specificity and stability are good, can be used for the clone of slow virus carrier integration site in complicated or trace sample, especially clinical sample and preclinical models sample.It is captured after obtaining integration site sequence by the means of PCR, then carries out positioning analysis in the genome with integration site of the bioinformatics method to capture, safety evaluatio can be carried out to the integration that slow virus carrier occurs in the genome of host cell.
Description
Technical field
The present invention relates to gene engineering technology fields, and in particular to a kind of integration of slow virus carrier in CAR-T cell
Locus Analysis in Shoots method, and the primer sequence for this method.
Background technique
Slow virus (HIV-1, HIV-2) is one kind in retrovirus, and existing slow virus carrier derives from multiple objects
Kind, including human immunodeficiency virus (human immunodeficiency virus, HIV), monkey immunodeficiency virus
(simian immunodeficiency virus, SIV) etc..It is based on lentiviral gene group, remove its express it is non-must
The gene needed, instead therapeutic gene is built-up.Slow virus is integrated on the chromosome of host cell can be steady in a long-term
Expression, and compared with other retrovirus (such as adenovirus), slow virus can not only infect division cells, and can also
The cell of nondividing phase is infected, the above advantage is based on, slow virus carrier (lentiviral vector) is shifted as foreign gene
Carrier is widely used in clinical gene therapy.
CAR-T refers to Chimeric antigen receptor (chimeric antigen receptor, CAR) T cell, is by chimeric antigen
Receptor imports in T cell, and T cell signaling zone is simultaneously combined in the variable region (scFv) of T cell expression single chain antibody, can regulate and control T
The function and specificity of cell, target killing tumor cell.First the T cell of patient itself is separated, is added packaged
Slow-virus infection T cell, slow virus carry CAR and enter T cell, and T cell is made to pass through chimeric antigen modification in vitro, after amplification,
Again it is transfused back patient's body, specific recognition target antigen is to kill target cell.
Gene transfering efficiency height, integration rate are also apparently higher than other genes and turn being clearly advantageous that using slow virus as carrier
Shifting method.However, slow virus carrier still cannot at present since integration site is one of the factor for determining foreign gene expression levels
Target gene is inserted into specific site but radom insertion, thus the possibility risk of the gene transfer of lentivirus-mediated it
First is that causing insertion mutation (insertional in its proto-oncogene that may be integrated into host cell or tumor suppressor gene
Mutagenesis), lead to the activation of oncogene or the inhibition of tumor suppressor gene, thus the generation of induced cancer.These are potential secondary
Effect has caused the very big concern of people, so we need the safety to the gene transfer of lentivirus-mediated to carry out
Evaluation.
If the integration site regularity of distribution of the clear slow virus carrier of energy within the scope of host cell full-length genome, can enrich
Slow virus carrier tendentious understanding of integration site in the genome of host cell, and its safety can be evaluated.
Summary of the invention
It is an object of the invention to solve it is how sensitiveer, special, stably obtain in CAR-T cell exogenous sequences and insert
The problem of entering the specific site into genome.
To achieve the goals above, we capture to obtain integration site sequence by the means of PCR, then use bioinformatics
Method positioning analysis.And then provide a kind of integration site analysis method of slow virus carrier in CAR-T cell.
Integration site analysis method of the slow virus carrier in CAR-T cell, comprising the following steps:
(1) specificity amplification primer LTRI, 5 ' the ends biotin mark of LTRI are designed according to the 5 ' area LTR of slow virus carrier
Note；Using CAR-T genomic DNA as template, make unidirectional linearity PCR amplification, collects amplified production with strepavidin magnetic beads；
(2) one 5 ' end phosphorylation is designed, the single stranded DNA linker (ssLC) of 3 ' end double deoxidations will be single with RNA ligase
5 ' the ends of chain DNA linker and the amplified production 3 ' of step (1) are held and are connected；
(3) it first round PCR amplification: holds the area LTR to design first pair according to DNAlinker sequence and slow virus carrier 5 ' and draws
Object, upstream primer LTRII, downstream primer LCI carry out PCR amplification by template of the connection product of step (2)；
(4) second wheel PCR amplifications: it holds the area LTR to design second pair according to DNAlinker sequence and slow virus carrier 5 ' and draws
Object, upstream primer LTRIII, downstream primer LCII carry out PCR amplification by template of the amplified production of step (3)；
(5) amplified production of step (4) carries out library construction, and sequencing analysis, you can get it slow virus carrier is in CAR-T
Integration site in cell.
The working principle of the above method is: first with the known array on Insert Fragment, the i.e. 5 ' area LTR of slow virus carrier
Design primer LTRI, using the CAR-T cell genomic dna of extraction as template, which is held along the 5 ' of genomic DNA to 3 ' ends
Linear amplification, and pass through the junction (junction) of carrier and genome；Due to being unidirectional primer, so amplified production is
Single-stranded DNA sequence different in size, and 5 ' ends of product are marked with biotin, so can be by being connected with strepavidin magnetic beads for line
Property amplified production is collected.
3 ' ends of linear PCR amplified production are unknown, to analyze it relatively difficult, therefore the present invention devises a list again
Chain DNA linker (ssLC), to its 5 ' end phosphorylation and 3 ' end double deoxidation (dideoxycytidine, 2'3'-ddc) modifications.
The purpose of 5 ' end phosphorylations is to connect the end DNA linker (ssLC) 5 ' and 3 ' ends of linear PCR amplified production by phosphorylation
It connects.The purpose of 3 ' end double deoxidation modifications is to prevent it from carrying out meaningless lasting extension amplification.By the 3 ' of linear PCR amplified production
After end connects upper DNAlinker (ssLC), sequence length is just secured.
Next, carrying out design of primers for these sequences, i.e. upstream primer LTRII (matches with 5 ' ends of connection product
It is right), downstream primer LCI (is matched) with 3 ' ends of connection product, is carried out PCR amplification by template of connection product, after annealing, is obtained
Obtain double-strand connection product DNA fragmentation.
Upstream primer LTRIII (matching with 5 ' ends of connection product) is redesigned, downstream primer LCII is (with connection product
3 ' end pairings), using gained double-strand connection product DNA fragmentation as template, exponential amplification is carried out, increases double-strand quantity, convenient for subsequent
Library is constructed, by library construction and sequencing analysis, final you can get it integration position of the slow virus carrier in CAR-T cell
Point.
Optional or preferred, in the above method, the CAR-T cell is anti-BCMACAR-T cell.
Optional or preferred, in the above method, the nucleotide sequence of the single stranded DNA linker (ssLC) is as follows: 5 '-
Cctaactgctgtgccactgaattcagatctcccgggtc-3 ' (SEQ ID NO:6), 5 ' ends carry out phosphorylation modification,
3 ' ends carry out double deoxidation modification.
The present invention also provides the primer for above-mentioned analysis method, nucleotide sequence is as follows:
LTRI:5 '-aggctcagatctggtctaaccag-3 ' (SEQ ID NO:1),
LCI:5 '-gacccgggagatctgaattcagtg-3 ' (SEQ ID NO:2),
LTRII:5 '-ggctcgccactccccagtcc-3 ' (SEQ ID NO:3),
LCII:5 '-gatctgaattcagtggcacagcagt-3 ' (SEQ ID NO:4),
LTRIII:5 '-tggcctcttctaccttatctgg-3 ' (SEQ ID NO:5).
Compared with prior art, the invention has the following advantages:
This method is independent of restriction enzyme, so not having non-specific amplification or endonuclease bamhi is too long causes to expand
The problems such as Increasing Efficiency declines.
Method high sensitivity, specificity and stability of the invention is good, can be used for slow virus in complicated or trace sample and carries
The clone of body integration site, especially clinical sample and preclinical models sample, provide for the safety evaluation of integration site
It ensures.
Detailed description of the invention
Fig. 1 is the slow virus carrier integration site electrophoretic analysis of different analyte captures in sample message table in embodiment
Figure.
Fig. 2 is the distribution of slow virus integration site on chromosome in anti-BCMACAR-T cell in embodiment.And base
Because between and intragenic distribution
Fig. 3 be in embodiment in anti-BCMACAR-T cell slow virus integration site between gene and intragenic point
Cloth.
Specific embodiment
The present invention is further explained in the light of specific embodiments, so that those skilled in the art can be better
Understand the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
Integration site analysis of 1 slow virus carrier of embodiment in CAR-T cell
Primer sequence see the table below:
1 experimental method
The extraction of 1.1anti-BCMACAR-T cellular genome total DNA
(1) sample homogenization
1ml DNAZOL Reagent is added into CAR-T cell sample, is blown and beaten 50 times with pipettor, pays attention to avoiding bubble
It generates.
(2) DNA is precipitated
100% ethyl alcohol of 0.5ml is added into above-mentioned cell homogenates liquid, turns upside down 10 times, is stored at room temperature 1~3min, it can
See floccule mass.15000g is centrifuged 5min.Supernatant is removed with pipettor.
(3) DNA is washed
75% ethyl alcohol of 1ml is added, turns upside down 10 times, 12000g is centrifuged 1min.Carefully supernatant is removed with pipettor.
It is repeated once.The lid of EP pipe is opened into 3~5min, is air-dried in air.
(4) DNA dissolves
The 8mM NaOH of suitable volumes is added according to precipitating size, stands overnight (4 DEG C).Added according to NaOH volume within second day
Enter 0.1M HEPES, adjusts pH to 8.4.Soft piping and druming mixes.
(5) measurement of DNA concentration
2 μ l samples are taken, the concentration for extracting DNA is measured with nucleic acid quantification instrument.
The capture of 1.2 integration sites
1.2.1 linear PCR
(1) it is directed to slow virus carrier (BCMA-CAR plasmid) 5 ' area LTR, design primer LTRI carries out linear PCR.Reference
The operation of Invitrogen company, U.S. Platinum Taq DNA exo+ polymerase specification.Establish 50 μ l PCR reactants
System:
(2) PCR amplification is carried out by following procedure
1.2.2Microcon-50 concentration
(3) above-mentioned 50 μ l PCR product is added in 450 μ l water, is transferred completely into the Filter of Microcon-50
In device, 15000g, be centrifuged 12min, 22 DEG C；
(4) overturning Filter device be placed in new collection tube, 1000g be centrifuged 3min, 22 DEG C；
(5) liquid after concentration is transferred completely into 1.5ml EP pipe, moisturizing adjusts volume to 50 μ l.
1.2.3 magnetic capture
(6) it takes 20 μ l strepavidin magnetic beads (Streptavidin Magnetic Beads) to be placed in magnetic frame, uses and move after 60s
Liquid device is sucked out supernatant and discards；
(7) 40 μ l PBS-0.1%BSA are added into magnetic bead, dispel resuspension, is placed in magnetic frame, is inhaled after 60s with pipettor
Supernatant discards out；It is repeated once；
(8) 20 μ l 3M LiCl are added into magnetic bead, dispel resuspension, are placed in magnetic frame, supernatant is sucked out with pipettor after 60s
It discards；
(9) 50 μ l 6M LiCl are added into magnetic bead, dispel resuspension；
(10) magnetic bead after transfer is resuspended, which is added in 50 μ l linear PCR products, (to be noted: suspension containing magnetic beads and PCR product body
Product is than being 1:1)；
(11) it is placed in roller bearing blending instrument (600rpm) mixing, is incubated at room temperature 12~18h.At this point, biotinylated PCR product
It is coupled with magnetic bead, referred to as DNA-beads compound；
(12) previous step DNA-beads compound is placed in magnetic frame, supernatant is sucked out with pipettor after 60s and discards, and will
DNA-beads compound is resuspended in 100 μ l ddH2In O.
1.2.4 connecting single-stranded linker
(13) following 10 μ l coupled reaction system is established, DNA-beads compound is made to connect single stranded DNA linker (ssLC)
(14) DNA-beads compound is placed in magnetic frame, supernatant is sucked out with pipettor after 60s and discards, and by DNA-
Beads compound is resuspended in 10 μ l linked systems；
(15) roller bearing blending instrument (600rpm) is placed in mix, incubation at room temperature 16~for 24 hours；
(16) 90 μ l ofddH are added2In the above-mentioned coupled reaction system of O, it is placed in magnetic frame, is sucked out after 60s with pipettor
It discards clearly；
(17) DNA-beads compound is resuspended in 100 μ l ddH2O, is placed in magnetic frame, and supernatant is sucked out with pipettor after 60s
It discards；
(18) DNA-beads compound is resuspended in 10 μ l ddH2Sample is transferred to new 1.5ml tube by O.
1.2.5 first round PCR amplification
(19) matched with 5 '-biotinylated vector-specific primers (LCI, LTRII) of linker-and
Make following PCR reaction system
(20) setting of PCR program is as follows
(21) it takes 20 μ l strepavidin magnetic beads (Streptavidin Magnetic Beads) to be placed in magnetic frame, uses and move after 60s
Liquid device is sucked out supernatant and discards；
(22) 40 μ l PBS-BSA are added into magnetic bead, dispel resuspension, is placed in magnetic frame, is sucked out after 60s with pipettor
It discards clearly；It is repeated once；
(23) 20 μ l 3M LiCl are added into magnetic bead, dispel resuspension, is placed in magnetic frame, is sucked out after 60s with pipettor
It discards clearly；
(24) 20 μ l 6M LiCl are added into magnetic bead, dispel resuspension；Add 20 μ l in the PCR product of every part of sample；
(25) into 20 μ l PCR products, (note: suspension containing magnetic beads are the magnetic bead after transfer is resuspended with PCR product volume ratio
1:1)；
(26) it is placed in roller bearing blending instrument (600rpm) mixing, is incubated at room temperature 12~18h；
(27) add 60 μ l ddH2O to DNA-beads compound；
(28) DNA-beads compound is placed in magnetic frame, supernatant is sucked out with pipettor after 60s and discards；Magnetic bead is resuspended
In 100 μ l ddH2In O, it is repeated once；
(29) DNA-beads compound is placed in magnetic frame, supernatant is sucked out with pipettor after 60s and discards；Magnetic bead is resuspended
In 10 μ l 0.1N NaOH (0.1N NaOH, that is, 0.1M NaOH, each diluted fresh)；
(30) it is placed in roller bearing blending instrument (600rpm) mixing, is incubated at room temperature 15min.
(31) above-mentioned sample is placed in magnetic frame, with pipettor supernatant is sucked out after 60s and is transferred to new EP and manages, at this time DNA with
Beads enrichment comes.
1.2.6 the second wheel PCR amplification
(32) it is formulated as follows reaction system
(33) setting of PCR program is as follows
1.2.7 PCR product purifies
(34) isometric phenol is added: chloroform: isoamyl alcohol (25:24:1) turns upside down several times, 14000rpm centrifugation,
10min, 4 DEG C；
(35) transfer supernatant (water phase) is managed to new 1.5ml EP；
(36) 1/10 volume 3M NaAc (pH5.2) is added, turns upside down and mixes well several times；
(37) the ice ethyl alcohol of two volumes is added afterwards, is mixed well again after mixing, is placed in -20 DEG C of precipitates overnights；
14000rpm centrifugation in (38) second days, 10min, 4 DEG C；
(39) DNA precipitating twice is washed with 75% ethyl alcohol, places 2~3min in air, evaporates into residual liquid dry；
(40) the water dissolving DNA precipitating of suitable volumes is added, surveys concentration with nucleic acid quantification instrument and carries out electrophoresis detection.
1.3 PCR product high-flux sequences and data are analyzed
Above-mentioned PCR product after purification is sent to Suzhou Jin Weizhi Biotechnology Co., Ltd, Illumina is used
Miseq platform is sequenced, and carries out bioinformatic analysis for high-flux sequence the data obtained.
1.3.1 PCR product library construction
(1) to sending survey PCR fragment to carry out end-filling reparation, cohesive end is converted in 3 ' end additions base " A ".
(2) DNA connector is added in cohesive end two sides by base complementrity.
(3) PCR amplification adds Index in target fragment end, completes the building and quality inspection of sequencing library.
1.3.2 upper machine sequencing
(4) sequencing library is integrated on sequence testing chip by bridge-type PCR, upper machine sequencing.
1.3.3 analysis of biological information
Sequencing is completed after obtaining the sequencing enough initial data of depth, that is, starts to carry out analysis of biological information, process master
To include following three phases:
(5) sequencing data is assessed: sequencing quality assessment is that enough satisfaction analyses require to evaluate the quality of data, the number after Quality Control
According to for comparing, and the quality of data is assessed, next step analysis can be carried out by reaching requesting party.
(6) read1 of Clean reads and read2 is merged according to overlap sequence, is generated single
Reads, and count.
(7) combined reads is compared with people with the genome in the area LTR of anti-BCMACAR plasmid, and counted
Comparison result.
(8) integration site is detected according to comparison result, and integration site is counted and analyzed.
2 sample detection results
Sample message
| Sample number into spectrum | Sample type | Quantity (a) |
| 1 | Water | |
| 2 | Normal person's VOL6T cell | 1×107 |
| 3 | Myelomatosis multiplex people's TMLT cell | 1×107 |
| 4 | Myelomatosis multiplex people TML turns BCMACAR-T cell | 1×107 |
| 5 | Myelomatosis multiplex people ZGL turns BCMACAR-T cell | 1×107 |
| 6 | Myelomatosis multiplex people CYB turns BCMACAR-T cell | 1×107 |
| 7 | Myelomatosis multiplex people TY turns BCMACAR-T cell | 1×107 |
| 8 | Myelomatosis multiplex people CXL turns BCMACAR-T cell | 1×107 |
| 9 | Normal person ZJ turns BCMACAR-T cell | 1×107 |
| 10 | Normal person VOL5 turns BCMACAR-T cell | 1×107 |
| 11 | Normal person VOL6 turns BCMACAR-T cell | 1×107 |
| 12 | Normal person VOL7 turns BCMACAR-T cell | 1×107 |
It is the slow virus carrier integration site that different samples use the above method to capture in sample message table referring to Fig. 1.
After the completion of high-flux sequence, the sequencing enough initial data of depth are obtained, that is, start to carry out analysis of biological information, it will
Spliced reads is compared with LTR and human genome.Statistics while the reads number compared to human genome and LTR sequence
Mesh, i.e. Chromic reads, are shown in Table 2
Slow virus integration site is shown in intragenic distribution on chromosome and between gene in anti-BCMA CAR-T cell
Fig. 2 and Fig. 3.
The above results show that slow virus carrier is integrated into CAR-T cellular genome without tendentiousness and Preference, will not integrate
To the proto-oncogene or tumor suppressor gene of host cell, lead to the activation of oncogene or the inhibition of tumor suppressor gene, it will not induced cancer
Generation.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection scope of the present invention
It is without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, of the invention
Within protection scope.Protection scope of the present invention is subject to claims.
Sequence table
<110>Wuhan wave is farsighted reaches Biotechnology Co., Ltd
<120>integration site analysis method and primer of the slow virus carrier in CAR-T cell
<160> 6
<170> SIPOSequenceListing 1.0
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<211> 23
<212> DNA
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<400> 1
aggctcagat ctggtctaac cag 23
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gacccgggag atctgaattc agtg 24
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ggctcgccac tccccagtcc 20
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gatctgaatt cagtggcaca gcagt 25
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tggcctcttc taccttatct gg 22
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<212> DNA
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ctaactgctg tgccactgaa ttcagatctc ccgggtc 37
Claims (4)
1. integration site analysis method of the slow virus carrier in CAR-T cell, which comprises the following steps:
(1) specificity amplification primer LTRI is designed according to the 5 ' area LTR of slow virus carrier, biotin labeling is used at the 5 ' ends of LTRI；With
CAR-T genomic DNA is template, makees unidirectional linearity PCR amplification, collects amplified production with strepavidin magnetic beads；
(2) one 5 ' end phosphorylation is designed, the single stranded DNA linker (ssLC) of 3 ' end double deoxidations will be single-stranded with RNA ligase
5 ' the ends of DNAlinker and the amplified production 3 ' of step (1) are held and are connected；
(3) first round PCR amplification: holding the area LTR to design pair of primers according to DNAlinker sequence and slow virus carrier 5 ', on
Primer LTRII, downstream primer LCI are swum, carries out PCR amplification by template of the connection product of step (2)；
(4) second wheel PCR amplifications: holding the area LTR to design second pair of primer according to DNAlinker sequence and slow virus carrier 5 ', on
Primer LTRIII, downstream primer LCII are swum, carries out PCR amplification by template of the amplified production of step (3)；
(5) amplified production of step (4) carries out library construction, and sequencing analysis, you can get it slow virus carrier is in CAR-T cell
Integration site.
2. analysis method according to claim 1, which is characterized in that the CAR-T cell is that anti-BCMACAR-T is thin
Born of the same parents.
3. analysis method according to claim 2, which is characterized in that the nucleotide of the single stranded DNA linker (ssLC)
Sequence is as follows: 5 '-cctaactgctgtgccactgaattcagatctcccgggtc-3 ', 5 ' end progress phosphorylation modifications, and 3 '
End carries out double deoxidation modification.
4. the primer for analysis method described in claim 3, which is characterized in that nucleotide sequence is as follows:
LTRI:5 ' biotin-aggctcagatctggtctaaccag-3 '
LCI:5 '-gacccgggagatctgaattcagtg-3 '
LTRII:5 ' biotin-ggctcgccactccccagtcc-3 '
LCII:5 '-gatctgaattcagtggcacagcagt-3 '
LTRIII:5 '-tggcctcttctaccttatctgg-3 '.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113046835A (en) * | 2019-12-27 | 2021-06-29 | 深圳华大生命科学研究院 | Sequencing library construction method for detecting lentivirus insertion site and lentivirus insertion site detection method |
| CN116403647A (en) * | 2023-06-08 | 2023-07-07 | 上海精翰生物科技有限公司 | Biological information detection method for detecting slow virus integration site and application thereof |
| WO2023179766A1 (en) * | 2022-03-24 | 2023-09-28 | 南京传奇生物科技有限公司 | Method for preparing dna library and detecting retroviral integration site |
| WO2024152493A1 (en) * | 2023-01-17 | 2024-07-25 | 宁波熙宁检测技术有限公司 | Method for detecting lentivirus integration site and use thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1820081A (en) * | 2003-05-07 | 2006-08-16 | 宝生物工程株式会社 | Method of analyzing gene introduction site |
| CN1995384A (en) * | 2006-01-06 | 2007-07-11 | 王铸钢 | Quick and convenient authentication technology fro transgenic insert locus |
| US20120076763A1 (en) * | 2009-04-30 | 2012-03-29 | The Regents Of The University Of California | Combination anti-hiv vectors, targeting vectors, and methods of use |
| CN102766688A (en) * | 2012-04-17 | 2012-11-07 | 盛司潼 | Method for testing gene sequences |
| CN106086077A (en) * | 2016-07-05 | 2016-11-09 | 北京普瑞金科技有限公司 | The slow virus carrier prepared for CAR T and construction method thereof and application |
| CN106987585A (en) * | 2017-03-15 | 2017-07-28 | 深圳市海普洛斯生物科技有限公司 | A kind of single stranded DNA two generations sequencing library construction method for cfDNA |
| CN107557388A (en) * | 2017-07-26 | 2018-01-09 | 生研医药科技（武汉）有限公司 | A kind of slow virus carrier prepared for CAR T and its construction method and application |
-
2018
- 2018-12-19 CN CN201811557035.XA patent/CN109554447A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1820081A (en) * | 2003-05-07 | 2006-08-16 | 宝生物工程株式会社 | Method of analyzing gene introduction site |
| CN1995384A (en) * | 2006-01-06 | 2007-07-11 | 王铸钢 | Quick and convenient authentication technology fro transgenic insert locus |
| US20120076763A1 (en) * | 2009-04-30 | 2012-03-29 | The Regents Of The University Of California | Combination anti-hiv vectors, targeting vectors, and methods of use |
| CN102766688A (en) * | 2012-04-17 | 2012-11-07 | 盛司潼 | Method for testing gene sequences |
| CN106086077A (en) * | 2016-07-05 | 2016-11-09 | 北京普瑞金科技有限公司 | The slow virus carrier prepared for CAR T and construction method thereof and application |
| CN106987585A (en) * | 2017-03-15 | 2017-07-28 | 深圳市海普洛斯生物科技有限公司 | A kind of single stranded DNA two generations sequencing library construction method for cfDNA |
| CN107557388A (en) * | 2017-07-26 | 2018-01-09 | 生研医药科技（武汉）有限公司 | A kind of slow virus carrier prepared for CAR T and its construction method and application |
Non-Patent Citations (2)
| Title |
|---|
| 张俊等: "应用反向PCR克隆慢病毒介导的转基因小鼠整合位点序列", 《生物技术通讯》 * |
| 钱卫等: "慢病毒载体在人角质形成细胞基因组中的整合位点分析", 《第三军医大学学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113046835A (en) * | 2019-12-27 | 2021-06-29 | 深圳华大生命科学研究院 | Sequencing library construction method for detecting lentivirus insertion site and lentivirus insertion site detection method |
| WO2023179766A1 (en) * | 2022-03-24 | 2023-09-28 | 南京传奇生物科技有限公司 | Method for preparing dna library and detecting retroviral integration site |
| WO2024152493A1 (en) * | 2023-01-17 | 2024-07-25 | 宁波熙宁检测技术有限公司 | Method for detecting lentivirus integration site and use thereof |
| CN116403647A (en) * | 2023-06-08 | 2023-07-07 | 上海精翰生物科技有限公司 | Biological information detection method for detecting slow virus integration site and application thereof |
| CN116403647B (en) * | 2023-06-08 | 2023-08-15 | 上海精翰生物科技有限公司 | Biological information detection method for detecting slow virus integration site and application thereof |
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