CN105907721A - Pig intestinal endothelial cell line for stably expressing CaS9 protein - Google Patents

Pig intestinal endothelial cell line for stably expressing CaS9 protein Download PDF

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CN105907721A
CN105907721A CN201610322402.2A CN201610322402A CN105907721A CN 105907721 A CN105907721 A CN 105907721A CN 201610322402 A CN201610322402 A CN 201610322402A CN 105907721 A CN105907721 A CN 105907721A
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cell
cas9
ipec
cell line
culture
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陈庄
刘文华
蒋宗勇
陈中健
朱翠
张群洁
朱庆锋
吴秀菊
张卫娜
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Guangdong Academy Of Agricultural Sciences-Agricultural Biological Gene Research Center
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Guangdong Academy Of Agricultural Sciences-Agricultural Biological Gene Research Center
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Priority to PCT/CN2017/000247 priority patent/WO2017193606A1/en
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Abstract

The invention discloses a pig intestinal endothelial cell line for stably expressing a CaS9 protein. The pig intestinal endothelial cell line is characterized in that non-transformed intestinal endothelial cell line IPEC-J2 of a neonatal pig is taken as a host cell and is transfected with a lentiviral expression vector containing CaS9, and a transformant capable of stably expressing the CaS9 protein is obtained by screening of 20mu g/mL of Blasticidin. A construction method of the pig intestinal endothelial cell line comprises the following steps: firstly, constructing the lentiviral expression vector containing the CaS9; secondly, transforming an HEK293T cell; thirdly, transfecting an IPEC-J2 cell; fourthly, performing monoclonal screening. The pig intestinal endothelial cell line IPEC-J2-CaS9 disclosed by the invention can stably express the CaS9 protein, and has a growth curve identical with that of the non-transformed IPEC-J2 cell, and cell morphology identical with that of the IPEC-J2 cell. The pig intestinal endothelial cell line disclosed by the invention has important application value in aspects of researching the growth and differentiation of pig intestinal endothelial cells as well as dealing with the stimulation from exogenous substances.

Description

A kind of swine intestinal epithelium cells system stably expressing Cas9 albumen
Technical field
The present invention relates to build the swine intestinal epithelium cells system of stable expression Cas9 albumen, belong to cell engineering field.
Background technology
CRISPR-Cas9 is a kind of to phage genome or the adaptive immune system of horizontal transfer plasmid present in antibacterial. When foreign DNA invades antibacterial, CRISPR transcribes in site generation two kinds of precursor tiny RNA, i.e. crRNA and tracrRNA, with Shi Hecheng has PROTEIN C as9 of endonuclease activity.Owing to Cas9 can identify the PAM site in foreign DNA and crRNA 5 ' 20 bases of end can be with base pair complementarity in foreign DNA, and the nucleic acid-protein of crRNA, tracrRNA and Cas9 composition is multiple Zoarium can specifically cut foreign DNA.The CRISPR-Cas9 of bacterial origin or its change a little after form sgRNA (crRNA With tracrRNA chimera)-Cas9 can be transplanted in eukaryote and exercise a kind of can the Cobra venom endonuclease merit of Programmed Design Can, there is the DNA of double-strand otch (DSB) after the mechanism such as NHEJ or HDR are repaired, the gene at cleavage site just produces slotting Enter, lack or editor's effect such as replacement.CRISPR-Cas9 is possible not only to operate one or several gene, it is thus achieved that one Or the cell that is knocked of multiple gene or animal model, and genomic level can be expanded to, it is each that scale builds species The library that gene is knocked is for resolving cell or the individual life process of complexity.Compared with traditional gene knockout method, CRISPR-Cas9 will not introduce exogenous dna fragment, and the cell line obtained or animal individual are without exogenous dna fragment.By institute The sgRNA that need to study gene imports in the cell line stably expressing Cas9 gene, it is possible to obtain lack this gene (or multiple gene) Cell, provide new means for studying this gene (or multiple gene) function.Meanwhile, the specific gene disappearance obtained Genome editor's cell line is alternatively cultivated safe transgenic animal individuality and is provided nuclear transplantation material.
In intensive pig production produces, easily there is " the wean of degradation under dyspepsia, diarrhoea, production performance in early-weaned piglets Piglet irritability syndrome " phenomenon.Many scholars both domestic and external carry out mechanism with pig source intestinal cell system to this syndrome in trial The research of aspect.IPEC-J2 cell line derives from new life non-suckling pig jejunum, by Berschneider HM in 1989 fraction of the year Obtain from cultivating.Different with other conventional intestinal cell systems (such as Caco-2), this cell line is non-transformed cell system, energy Physiological environment in analogue body very well.This cell line can express the multiple cytokine relevant to innate immunity, and in energy analogue body, intestinal is thin Born of the same parents tackle produced innate immunity reaction when enteric microorganism source stimulates.Can according to culture medium not when cultivating in vitro With polarizing, can enterocyte phenotypic differentiation in analogue body well.Therefore, IPEC-J2 cell line is research newborn piglet Intestinal absorption and the fabulous external model of immunologic function.By knocking out the functional sequence of genome in IPEC-J2 cell line one by one, Obtaining the cell of functional trait to be studied, the mechanism for research regulation and control " ablactational baby pig irritability syndrome " provides extremely important material. But, the most not yet have the swine intestinal epithelium cells system that can stably express Cas9 albumen of commercialization.
Due to the similarity at aspects such as anatomical structure and digestion, absorption, physiological metabolisms, newborn piglet is considered as neonate The ideal animals model of nutrient research.Baby is closely similar with the intestinal growth situation of newborn piglet, therefore application genome editor Research ewborn infant intestinal self can be grown, to absorption of nutrient ingredients feelings by the swine intestinal epithelium cells system IPEC-J2-Cas9 crossed Condition, provides strong reference value to allogenic material response.
Summary of the invention
For above-mentioned prior art, the invention provides and build the swine intestinal epithelium cells system stably expressing Cas9 albumen IPEC-J2-Cas9).This cell line both may be used for the genome editor's cell line screened specific gene disappearance or insert, it is also possible to The signal path offer new tool interacted with intestinal for research specific gene function or parsing microorganism.
The present invention is achieved by the following technical solutions:
A kind of swine intestinal epithelium cells system stably expressing Cas9 albumen, is with newborn piglet non-transformed intestinal epithelial cell system IPEC-J2 is host cell, the transfection Lentiviral containing Cas9, screens through the Blasticidin of 20 μ g/mL The transformant that can stably express Cas9 albumen obtained.
The construction method of the described swine intestinal epithelium cells system stably expressing Cas9 albumen, comprises the following steps:
(1) structure of the Lentiviral containing Cas9:
SpCas9 gene, construction expression Cas9 gene and blasticidin S (Blasticidin) resistant gene is expanded from pX330 The fusion fragment of EFS:SpCas9:Flag:P2A:Blast, insert slow virus carrier, it is thus achieved that the slow virus containing Cas9 is expressed and carries Body LentiCas9-Blast；Slow virus packaging system is collectively constituted with packaging plasmid psPAX2 and envelope plasmid pMD2.G；
(2) HEK293T cell is converted:
Above-mentioned slow virus packaging system converts HEK293T cell, in 37 DEG C, 5%CO2Cell culture incubator is cultivated；After 24h, Changing DMEM/F12 complete medium into, continue to cultivate 24～48h, collection culture fluid is in centrifuge tube, centrifugal, obtains slow virus Supernatant, is dispensed into cell cryopreservation tube, and-80 DEG C of Refrigerator stores are standby；
The method of concrete conversion HEK293T cell is:
By 7 × 105HEK293T cell is inoculated in Tissue Culture Dish, 37 DEG C, 5%CO2In incubator overnight (10～14h) Cultivate, wait growing to 60～during 80% degrees of fusion, obtain HEK293T cell culture fluid, standby；
Take two 1.5mL centrifuge tubes, one of them centrifuge tube adds 20 μ LSerum-free concentration cultures, 1 μ g LentiCas9-Blast plasmid, 750ng psPAX2 plasmid and 250ng pMD2.G plasmid, mixing, room temperature stands 5min； Another one centrifuge tube adds 74uLSerum-free concentration cultures, 6uLHD transfection reagent；Two Pipe mixes, and incubation at room temperature 20min adds above-mentioned HEK293T cell culture fluid, and mixing, in 37 DEG C, 5%CO2Cell Incubator is cultivated；After 24h, change DMEM/F12 complete medium into, continue to cultivate 24～48h, collect culture fluid in 15 In mL centrifuge tube, centrifugal, take supernatant and be dispensed into cell cryopreservation tube ,-80 DEG C of Refrigerator stores；
(3) transfection IPEC-J2 cell:
Cultivation IPEC-J2 cell, to degrees of fusion 70～80%, is subsequently adding slow virus supernatant, after slow-virus transfection 12h, changes Become fresh culture medium, continue at 37 DEG C, 5%CO2Constant temperature cell culture incubator is cultivated 12h；Subsequently, it is replaced with containing 20 μ g/mL The fresh culture of Blasticidin, 37 DEG C, 5%CO2Constant temperature cell culture incubator cultivates transfectional cell 5～7d, collects transfection Cell, standby；
(4) monoclonal screening:
Use limiting dilution assay to be diluted to 7～8 cell/mL the transfectional cell in above-mentioned (3) by culture medium, add 96-hole In each hole in plate so that the cell average in each hole is 1.4～1.6；Until unicellular grow up to cell mass after, move on to 12-orifice plate makees amplification culture, collects cell, i.e. obtain the swine intestinal epithelium cells system of stable expression Cas9 albumen, subpackage, It is saved in liquid nitrogen.
In above-mentioned steps, the most detailed description, the technology not specialized, it is existing routine techniques means in prior art, All can carry out according to conventional molecular biological and Cell Biology Experiment condition or the condition advised according to manufacturer's description.
The swine intestinal epithelium cells system IPEC-J2-Cas9 of the stable expression Cas9 albumen of the present invention, it is possible to stably express Cas9 egg In vain, growth curve is identical with unconverted IPEC-J2 cell, and cellular morphology is identical with IPEC-J2 cell.
The swine intestinal epithelium cells system IPEC-J2-Cas9 of the stable expression Cas9 albumen of the present invention, may be used for tying with sgRNA Genome editor's cell line that conjunction structure specific gene (functional sequence) or multiple gene (functional sequence) knock out, is used for studying It is knocked cytology's function of gene (functional sequence), at research intestine of young pigs epithelial cell growth, breaks up and tackle external source Material incentive aspect has important using value.
The cell line of the present invention, both may be used for the genome editor's cell line screened specific gene disappearance or insert, it is also possible to for The signal path offer new tool that research specific gene function or parsing microorganism interact with intestinal, has filled up and has not yet had at present May be used for the blank of the cell line of the IPEC-J2-Cas9 of genome editor.The cell line of the present invention, has the advantages that
1) cell line IPEC-J2-Cas9 not yet having commercialization to can be used for pig genome editor is provided；
2) this cell line applied basic research of single or multiple genes of interest in may be used for knock-out pig genome,
3) this cell line can obtain the stable cell lines of single or multiple gene knockout；
4) intestinal epithelial cell under this cell line can simulate normal physiological condition, is produced any environmental stimuli by research intestinal Raw physiologically stress provides effective external model, such as resolving the letter that microorganism interacts with swine intestinal epithelium cells Number path.
Accompanying drawing explanation
Fig. 1: the PCR testing result of slow virus packaging system plasmid.
Fig. 2: the collection of illustrative plates of slow virus packaging system plasmid, wherein, the collection of illustrative plates of A:LentiCas9-Blast；B:psPAX2's Collection of illustrative plates；The collection of illustrative plates of C:pMD2.G.
The titer determination of Fig. 3: Cas9 gene packaging virus, wherein, T sample A=3.25 × 108；T sample B=3.95 × 107。
Fig. 4: limiting dilution assay obtains cell growth status during monoclonal cell system, wherein, A: individual cells (2d)； B: several cells (6d)；C: cell clone group (9d).
The insertion in IPEC-J2-Cas9 cell line genome of Fig. 5: the Cas9 gene.
The expression in IPEC-J2-Cas9 cell line of Fig. 6: the Cas9 gene.
The growth curve of Fig. 7: IPEC-J2 and IPEC-J2-Cas9 cell line.
Fig. 8: detect IPEC-J2 and IPEC-J2-Cas9 cellular morphology, wherein, A:IPEC-J2 with hematoxylin-eosin staining Cell；B:IPEC-J2-Cas9 cell.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated.
Instrument involved in following embodiment, reagent, material etc., unless otherwise noted, be in prior art existing often Rule instrument, reagent, material etc., can be either commercially available by regular.Experimental technique involved in following embodiment, detection Methods etc., unless otherwise noted, are existing normal experiment method, detection method etc. in prior art.
(1) structure of the Lentiviral containing Cas9:
SpCas9 gene, construction expression Cas9 gene and blasticidin S is expanded from pX330 (Addgene#42230) (Blasticidin) the fusion fragment of the EFS:SpCas9:Flag:P2A:Blast of resistant gene, inserts slow virus carrier, obtains The Lentiviral LentiCas9-Blast (as shown in Figure 1) of Cas9 must be contained；With the packaging plasmid psPAX2 bought (Addgene#12260) and envelope plasmid pMD2.G (Addgene#12259) collectively constitute slow virus packaging system (as Fig. 2 A, Shown in B, C).
(2) HEK293T cell is converted:
By 7 × 105HEK293T cell is inoculated in Tissue Culture Dish, 37 DEG C, 5%CO2Incubated overnight in incubator, waits to grow to 60%～during 80% degrees of fusion, obtain HEK293T cell culture fluid, standby；
Take two 1.5mL centrifuge tubes, one of them centrifuge tube adds 20 μ LSerum-free concentration cultures, 1 μ g LentiCas9-Blast plasmid, 750ng psPAX2 plasmid and 250ng pMD2.G plasmid, mixing, room temperature stands 5min； Another one centrifuge tube adds 74 μ LSerum-free concentration cultures, 6 μ LHD transfection reagent；Two Pipe mixes, and incubation at room temperature 20min adds above-mentioned HEK293T cell culture fluid, and mixing, in 37 DEG C, 5%CO2Cell Incubator is cultivated；After 24h, change DMEM/F12 complete medium into, continue to cultivate 24～48h, collect culture fluid in 15 In mL centrifuge tube, centrifugal, take supernatant and be dispensed into cell cryopreservation tube ,-80 DEG C of Refrigerator stores.
(3) titre of mensuration virion:
Take out the 150 μ L culture supernatant containing slow virus, utilizeRNA Virus Kit extracts slow virus base Because of group RNA；Use Quant-XTM One-Step qRT-PCRKit at 42 DEG C, 5min；95 DEG C, 10sec condition Lower RNA reverse transcription is become cDNA；On quantitative real time PCR Instrument, carry out 95 DEG C of 40 circulations, 5sec；60 DEG C, 30sec PCR reaction；Then at 95 DEG C, 15sec；60 DEG C, renaturation of dissociating under the conditions of 30sec obtains dissociation curve；Employing standard Curve method measures the starting copy number of RNA in reaction system；Virus genome RNA copy number is calculated: virus is dripped further according to formula Degree=[(RNA initial quantity (RNA initial amount)) (100 μ L/mL) (50 μ L elution)]/[(150 μ L Sample) (2 μ L added to well) (2copies of RNA/viral particle)] (as shown in Figure 3).Result: Slow virus successful conversion HEK293T cell, and obtain titre and be respectively 3.25 × 108With 3.95 × 107Virion, available In following transfection IPEC-J2 cell.
(4) IPEC-J2 cell drug sensitivity was examined:
The IPEC-J2 digestion of exponential phase will be cultivated, and collect cell and count；In one piece of 96 orifice plate, every hole is inoculated 1500 IPEC-J2 cells, 37 DEG C, 5%CO2Overnight incubation；When covering the 80% of hole floor space Deng cellular layer, use pipettor Remove supernatant, add the Blasticidin (1～55 μ g/mL) of serial dilutions；The Blasticidin of each concentration Do 4 repeating holes, observe the cell survival in each hole every day；Record specific Blasticidin in the 7th to the tenth day dense The situation that the lower cell of degree is the most dead in 4 repeating holes, selecting minimum Blasticidin concentration is sensitivity experiments Sensitive concentration be 20 μ g/mL, express the IPEC-J2-Cas9 cell monoclonal of Cas9 for screening in following (6). Result: this experiment obtains the antibiotic sensitive concentration for screening IPEC-J2-Cas9 cell line.
(5) transfection IPEC-J2 cell:
Incubated overnight length to degrees of fusion 70～80% IPEC-J2 cell for virus transfection；According to the slow virus supernatant recorded Titre value, is added the slow virus supernatant of proper volume by infection multiplicity (MOI) 0.2～0.3, after slow-virus transfection 12h, Change fresh culture into, continue at 37 DEG C, 5%CO2Constant temperature cell culture incubator is cultivated 12h；Subsequently, it is replaced with containing 20 μ g/mL The fresh culture of Blasticidin, 37 DEG C, 5%CO2Constant temperature cell culture incubator selects transfectional cell 5～7d, collects transfection Cell screens single cell clone for limiting dilution assay.
(6) monoclonal screening:
By culture medium, the transfectional cell in above-mentioned (5) is diluted to 7～8 cell/mL, takes 200 μ L and add in 96-orifice plate Each hole in so that the cell average in each hole is 1.4～1.6；Under the microscope, confirm the hole containing individual cells, The growing state (as shown in Figure 4) of observation of cell.Until unicellular grow up to cell mass after, move on to 12-orifice plate makees amplification culture, Collect cell, subpackage, be saved in liquid nitrogen.Result: obtained the IPEC-J2 monoclonal containing Cas9 gene.
(7) Cas9 gene insertion situation detection:
Extract IPEC-J2-Cas9 cell line genomic DNA, do negative control with IPEC-J2 cell line genomic DNA, LentiCas9-Blast plasmid does positive control, uses primer Cas9F:5 '-ATG GAC AAG AAG TAC AGC ATC GGC CTG-3 ' and primer Cas9R:5 '-GTT CAG GTC GCC CTC GAT CAG GAA GTG-3 ' PCR augmentation detection Whether IPEC-J2-Cas9 genome exists about 500bp fragment (as shown in Figure 5).Result: the IPEC-J2-Cas9 of acquisition Monoclonal cell system genome contains Cas9 gene.
(8) Cas9 protein expression detection:
Extract IPEC-J2-Cas9 cell line total protein with RIPA Buffer, measure the concentration of albumen with Qubit 2.0.Preparation The SDS-PAGE glue of 10%, the albumen adding about 100 μ g carries out electrophoresis.After electrophoresis, use half-dried transfer printing transferring film.With Pierce Clear Milk Blocking Buffer room temperature jog closes 60min, the anti-Monoclonal ANTI-FLAG that adds M2antibody produced in mouse, 4 DEG C of jog night incubation.After washing film with the TBS containing 0.05%Tween-20, Anti-Anti-Mouse IgG (the whole-molecule)-Peroxidase that adds two, room temperature jog hatches 60min.It is unnecessary to wash Antibody after, with SuperSignal west pico chemiluminescent substrate (ECL) chemical illuminating reagent Box colour developing (Fig. 6).Result: Cas9 albumen is stably expressed by the IPEC-J2-Cas9 monoclonal cell system of acquisition.
(9) the IPEC-J2-Cas9 cell growth curve stably expressing Cas9 measures:
Taking the cell that growth conditions is good, PBS rinses cell surface 3 times, uses trypsinization 5min, makes with fresh culture Count after cell suspension.According to cell counts, make Secondary Culture according to 10000/mL, every hole 1mL, connect 12 orifice plates, Every strain cell connects 36 holes.Start counting up cell after 24h, count once every 24h later, take three porocytes every time, point Do not calculate average.Continuous counter 10d.According to cell counts, with cell quantity as vertical coordinate, with the time (d) for horizontal Coordinate draws growth curve (Fig. 7).Result: the growth curve of the IPEC-J2-Cas9 monoclonal cell of acquisition and unconverted IPEC-J2 cell is identical.
(10) IPEC-J2-Cas9 morphocytology detection:
Hematoxylin-eosin staining detection cellular morphology: by 2 × 105The IPEC-J2 cell of/mL and IPEC-J2-Cas9 cell are respectively Cultivate on sterile cover slips, 37 DEG C, 5%CO2Overnight incubation.Suck the culture fluid in coverslip, rinse 3 times with PBS. After fixing cell by neutral formalin, add 0.25%Triton-100 and process cell 5min.After PBS rinses 3 times, add MAYER brazilwood extract dyeing liquid dyeing 10min, rinses coverslip to water colorless with water.Successively with at 75% ethanol, 85% ethanol Reason 2min, Yihong processes 30sec.95% Ethanol Treatment 2min, dehydrated alcohol processes 4min.Neutral tree is dripped in microscope slide Fat mounting, is just putting basis of microscopic observation cellular morphology take pictures (Fig. 8).Result: the IPEC-J2-Cas9 monoclonal cell of acquisition Cellular morphology identical with IPEC-J2 cell.
Conclusion: this experiment obtains a kind of IPEC-J2-Cas9 cell line stably expressing Cas9 albumen, the physiology of this cell line State is identical with unconverted IPEC-J2 cell, can be as the in vitro study model of the intestinal epithelial cell under physiological condition.

Claims (3)

1. the swine intestinal epithelium cells system stably expressing Cas9 albumen, it is characterized in that: be with newborn piglet non-transformed intestinal epithelial cell system IPEC-J2 as host cell, the transfection Lentiviral containing Cas9, the blasticidin S (Blasticidin) through 20 μ g/mL screens the transformant that can stably express Cas9 albumen obtained.
2. the construction method of the swine intestinal epithelium cells system of the stable expression Cas9 albumen described in claim 1, it is characterised in that: comprise the following steps:
(1) structure of the Lentiviral containing Cas9:
Expand SpCas9 gene, the fusion fragment of the EFS:SpCas9:Flag:P2A:Blast of construction expression Cas9 gene and Blasticidin resistant gene from pX330, insert slow virus carrier, it is thus achieved that the Lentiviral LentiCas9-Blast containing Cas9；Slow virus packaging system is collectively constituted with packaging plasmid psPAX2 and envelope plasmid pMD2.G；
(2) HEK293T cell is converted:
Above-mentioned slow virus packaging system converts HEK293T cell, in 37 DEG C, 5%CO2Cell culture incubator is cultivated；After 24h, changing DMEM/F12 complete medium into, continue to cultivate 24～48h, collection culture fluid is in centrifuge tube, centrifugal, obtains slow virus supernatant, standby；
(3) transfection IPEC-J2 cell:
Cultivation IPEC-J2 cell, to degrees of fusion 70%～80%, is subsequently adding slow virus supernatant, after slow-virus transfection 12h, changes fresh culture medium into, continues at 37 DEG C, 5%CO2Constant temperature cell culture incubator is cultivated 12h；Subsequently, it is replaced with the fresh culture containing 20 μ g/mL Blasticidin, 37 DEG C, 5%CO2Constant temperature cell culture incubator cultivates transfectional cell 5～7 days, collects transfectional cell, standby；
(4) monoclonal screening:
By culture medium, the transfectional cell in above-mentioned (5) is diluted to 7～8 cell/mL, adds in each hole of culture plate so that the cell average in each hole is 1.4～1.6；Until unicellular grow up to cell mass after, amplification culture, collect cell, i.e. obtain the swine intestinal epithelium cells system of stable expression Cas9 albumen.
Preparation method the most according to claim 2, it is characterised in that: in described step (2), the method for concrete conversion HEK293T cell is:
By 7 × 105HEK293T cell is inoculated in Tissue Culture Dish, 37 DEG C, 5%CO2Incubated overnight in incubator, wait growing to 60～during 80% degrees of fusion, obtains HEK293T cell culture fluid, standby；
Take two 1.5mL centrifuge tubes, one of them centrifuge tube adds 20 μ LSerum-free concentration cultures, 1 μ g LentiCas9-Blast plasmid, 750ng psPAX2 plasmid and 250ng pMD2.G plasmid, mixing, room temperature stands 5min；Another one centrifuge tube adds 74 μ LSerum-free concentration cultures, 6 μ LHD transfection reagent；Two pipe mixing, incubation at room temperature 20min, add above-mentioned HEK293T cell culture fluid, mixing, in 37 DEG C, 5%CO2Cell culture incubator is cultivated；After 24h, changing DMEM/F12 complete medium into, continue to cultivate 24～48h, collection culture fluid is in 15mL centrifuge tube, centrifugal, takes supernatant and is dispensed into cell cryopreservation tube ,-80 DEG C of Refrigerator stores.
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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2017193606A1 (en) * 2016-05-13 2017-11-16 广东省农业科学院农业生物基因研究中心 Porcine intestinal epithelial cell line stably expressing cas9 protein
CN107760652A (en) * 2017-09-29 2018-03-06 华南理工大学 The cell models of caco 2 and its method that CRISPR/CAS9 mediate drugs transporter target knocks out
CN108342416A (en) * 2018-02-08 2018-07-31 广州医科大学 A kind of conditionity induction knocks out the construction method for the liver cancer cell lines for being overexpressed Chd1l genes
CN112813031A (en) * 2021-01-14 2021-05-18 中国医学科学院基础医学研究所 Construction method and application of stable-transfer expression SpCas9 protein white cell line
CN113234684A (en) * 2021-05-10 2021-08-10 广东省农业科学院农业生物基因研究中心 LLC-PK1 cell line for stably expressing Cas9 protein and construction method thereof

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