Jump -In Parental Cell Lines Overview · Jump-In TM Parental Cell Line Kit Application Note Version...
6
Representative Data for Jump-In Parental Tel: 800 955 6288 x40266 ● E-ma Jum Overview Generating Isogenic Cells with Ju targeting technology based on R4 int allows the targeted integration of gene effort required for generation of stable c genomic locus provides the ideal solutio The Jump-In ™ Parental Cell Lines w “promoterless” Blasticidin resistance ge Positive clones containing stable geno resistance, picked by single-cell sorting genome by Southern blot and copy nu retargeting by transfection with the pJT selection with Blasticidin for 2 to 3 week Figure 1: Schem The Jump-In ™ Parental Cell Lines ca (generated from pJTI ™ R4 DEST CMV expressing the R4 Integrase (Figure 3 retargeting expression construct are site target site. This integration event a “promoterless” Blasticidin resistance g using Blasticidin (Figure 2). Jump-In TM Parental Cell Line Kit Application Note Version No. 1 n TM GripTite TM HEK 293 and CHO-K1 l Cell Lines ail: [email protected] ● Web: www.li mp-In TM Parental Cell Lines ump-In TM Parental Cell Lines. Jump-In™ cell enginee tegrase mediated site specific homologous recombinati etic material into a specific pre-engineered site, which by cell lines compared to standard methods. Isogenic expres on for comparative analysis of gene families, isotypes or o were generated by genomic integration of an R4 a ene (BSD) using homologous recombination via phiC31 i omic integrations were selected based on their newly a g, and then tested to determine the number of R4 sites pre number analysis. Clones with a single R4 integration site TI ™ R4 EXP CMV EmGFP pA and JTI ™ R4 Int vectors, f ks. matic representation of Jump-In™ Cell Line Generation an be retargeted by co-transfection with a retargeting e V pA using the Gateway ® cloning technology) and the 3, below). During retargeting, the genetic elements of int e-specifically integrated into the genome of the platform ce also positions the constitutive human EF1α promote gene, thus allowing the selection of successfully “retarg 1 Page 1 of 6 ifetechnologies.com ering is a novel gene ion. This technology y design reduces the ssion from a defined rthologs. acceptor site and a integrase (Figure 1). acquired Hygromycin esent in the host cell e were validated for followed by antibiotic expression construct pJTI ™ R4 Int vector terest carried by the ell line at the R4 attP er upstream of the geted” transformants
Transcript of Jump -In Parental Cell Lines Overview · Jump-In TM Parental Cell Line Kit Application Note Version...
Representative Data for Jump- In
Parental Cell Lines
Tel: 800 955 6288 x40266 ● E-mail:
Jump
Overview Generating Isogenic Cells with Jumptargeting technology based on R4 integraseallows the targeted integration of genetic material into a specific preeffort required for generation of stable cell lines compared to standard genomic locus provides the ideal solution for comparative analysis of gene families, isotypes or orthologs. The Jump-In™ Parental Cell Lines were generated by genomic integration of an R4 acceptor site and a “promoterless” Blasticidin resistance gene (BSD) using homologous recombination via phiC31 integrase (Figure 1). Positive clones containing stable genomic integrations were selected based on their newly acquired Hygromycin resistance, picked by single-cell sorting, and then tested to determine the number of R4 sites present in the host cell genome by Southern blot and copy number analysis. Clones with a single R4 integration site were validated for retargeting by transfection with the pJTIselection with Blasticidin for 2 to 3 weeks.
Figure 1: Schematic representation of Jump
The Jump-In™ Parental Cell Lines can be retargeted by co(generated from pJTI™ R4 DEST CMV pAexpressing the R4 Integrase (Figure 3, below). During retargeting, the genetic elements of interest carried by retargeting expression construct are sitetarget site. This integration event also positions the constitutive human EF1“promoterless” Blasticidin resistance gene, thus allowing the selection of successfully “retargeted” transformants using Blasticidin (Figure 2).
Jump-InTM Parental Cell Line Kit Application Note Version No. 1
InTM GripTite TM HEK 293 and CHO-K1 Parental Cell Lines
mail: [email protected] ● Web: www.lifetechnologies.com
Jump -InTM Parental Cell Lines
Jump-InTM Parental Cell Lines. Jump-In™ cell engineering is a novel gene targeting technology based on R4 integrase mediated site specific homologous recombination. This technology allows the targeted integration of genetic material into a specific pre-engineered site, which by design reduces the effort required for generation of stable cell lines compared to standard methods. Isogenic expression from a defined genomic locus provides the ideal solution for comparative analysis of gene families, isotypes or orthologs.
Parental Cell Lines were generated by genomic integration of an R4 acceptor site and a promoterless” Blasticidin resistance gene (BSD) using homologous recombination via phiC31 integrase (Figure 1).
Positive clones containing stable genomic integrations were selected based on their newly acquired Hygromycin sorting, and then tested to determine the number of R4 sites present in the host cell
genome by Southern blot and copy number analysis. Clones with a single R4 integration site were validated for retargeting by transfection with the pJTI™ R4 EXP CMV EmGFP pA and JTI™ R4 Int vectors, followed by antibiotic selection with Blasticidin for 2 to 3 weeks.
Schematic representation of Jump-In™ Cell Line Generation
Parental Cell Lines can be retargeted by co-transfection with a retargeting expression construct R4 DEST CMV pA using the Gateway® cloning technology) and the pJTI
expressing the R4 Integrase (Figure 3, below). During retargeting, the genetic elements of interest carried by retargeting expression construct are site-specifically integrated into the genome of the platform cell line at the R4 target site. This integration event also positions the constitutive human EF1α promoter upstream of the
n resistance gene, thus allowing the selection of successfully “retargeted” transformants
Version No. 1 Page 1 of 6
www.lifetechnologies.com
In™ cell engineering is a novel gene mediated site specific homologous recombination. This technology
engineered site, which by design reduces the methods. Isogenic expression from a defined
genomic locus provides the ideal solution for comparative analysis of gene families, isotypes or orthologs.
Parental Cell Lines were generated by genomic integration of an R4 acceptor site and a promoterless” Blasticidin resistance gene (BSD) using homologous recombination via phiC31 integrase (Figure 1).
Positive clones containing stable genomic integrations were selected based on their newly acquired Hygromycin sorting, and then tested to determine the number of R4 sites present in the host cell
genome by Southern blot and copy number analysis. Clones with a single R4 integration site were validated for R4 Int vectors, followed by antibiotic
with a retargeting expression construct cloning technology) and the pJTI™ R4 Int vector
expressing the R4 Integrase (Figure 3, below). During retargeting, the genetic elements of interest carried by the specifically integrated into the genome of the platform cell line at the R4 attP
promoter upstream of the n resistance gene, thus allowing the selection of successfully “retargeted” transformants
Representative Data for Jump- In
Parental Cell Lines
Tel: 800 955 6288 x40266 ● E-mail:
Figure 2: Targeted integration of the gene of interest (GOI) and a promoter for Blasticidin (BSD).
Figure 3: Workflow for the retargeting of
Advantages of using Jump
� Rapid and efficient generation of engineered cell l ines− Functional cell pools can be generated in as − No laborious clone isolation and analysis
ψψψψ
Jump-InTM Parental Cell Line Kit Application Note Version No. 1
InTM GripTite TM HEK 293 and CHO-K1 Parental Cell Lines
mail: [email protected] ● Web: www.lifetechnologies.com
Targeted integration of the gene of interest (GOI) and a promoter for Blasticidin (BSD).
orkflow for the retargeting of a Jump-In™ parental cell line with your gene of interest (GOI)
Advantages of using Jump -InTM Parental Cell Line Kits
Rapid and efficient generation of engineered cell l ines Functional cell pools can be generated in as little as 2-3 weeks No laborious clone isolation and analysis
R4 attP
BSD
Genomic target siteψψψψ ψψψψ
Promotor
R4 attB
attRBSD
ψψψψ
Integrated Construct
attL
BSD activated
GOI
GOI
Version No. 1 Page 2 of 6
www.lifetechnologies.com
Targeted integration of the gene of interest (GOI) and a promoter for Blasticidin (BSD).
In™ parental cell line with your gene of interest (GOI)
Representative Data for Jump- In
Parental Cell Lines
Tel: 800 955 6288 x40266 ● E-mail:
− Accepts large multi-gene inserts− Generate an unlimited number of cell lines without complicated licenses or restrictions to interpret
� Isogenic expression
− All cell lines derived from a given the same genomic locus.
− � Ideal solution for
− Cells and assays problematic for transient engineering technologies− Difficult to “engineer” cell lines− Expression of target panels (families/isoforms/
Figure 4: Time savings comparsion of Jump
InTM approach provides isogenic cells in approximately half the time as traditional clonal cell line engineering.
Table of Contents
− Validation of the Jump-InTM Parental Cell Lines to contain a single R4 site
− Stability of the Jump-InTM Parental Cell Lines
− Stability of the Jump-InTM retargeted
− Validation of retargeted cells
− Performance of retargeted cells in CellSensor
− Performance of retargeted cells in LanthaScreen
− Reproducibility of the retargeting
− GFP expression from retargeting with the control vector
Jump-InTM Parental Cell Line Kit Application Note Version No. 1
InTM GripTite TM HEK 293 and CHO-K1 Parental Cell Lines
mail: [email protected] ● Web: www.lifetechnologies.com
gene inserts Generate an unlimited number of cell lines without complicated licenses or restrictions to interpret
All cell lines derived from a given parental Jump-In™ cell line express the gene of interest from
Cells and assays problematic for transient engineering technologies Difficult to “engineer” cell lines Expression of target panels (families/isoforms/orthologs)
Time savings comparsion of Jump-InTM cell engineering vs standard cell engineering shows that the Jumpapproach provides isogenic cells in approximately half the time as traditional clonal cell line engineering.
Parental Cell Lines to contain a single R4 site
Parental Cell Lines
etargeted cells
Performance of retargeted cells in CellSensor® Reporter Gene Assays
Performance of retargeted cells in LanthaScreen® Assays
Reproducibility of the retargeting
GFP expression from retargeting with the control vector
Version No. 1 Page 3 of 6
www.lifetechnologies.com
Generate an unlimited number of cell lines without complicated licenses or restrictions to interpret
In™ cell line express the gene of interest from
cell engineering vs standard cell engineering shows that the Jump-approach provides isogenic cells in approximately half the time as traditional clonal cell line engineering.
Jump-InTM Parental Cell Line Kit Application Note Version No. 1 Page 4 of 6
Representative Data for Jump-In TM GripTite TM HEK 293 and CHO-K1 Parental Cell Lines
Tel: 800 955 6288 x40266 ● E-mail: [email protected] ● Web: www.lifetechnologies.com
Figure 5: Validation of the Jump-InTM Parental Cell Lines to contain a single R4 site
The number of genomic R4 sites was determined by Southern Blot using
genomic DNA from Jump-In™ GripTite™ 293 cells and Jump-InTM CHO-
K1 cells. One R4 site was found in both Jump-InTM Parental Cell Lines.
Figure 6: Stability of the Jump-InTM Parental Cell Lines
Jump-In™ HEK293 GripTite™ cells were propagated for 20 passage
before retargeting with GFP, GFP-PRAS40, GFP-p53 or NFkB beta
lactamase reporter and analyzed by fluorescent activated cell sorting
(FACS). The NFkB negative control showed no fluorescent green cells
and all of the other retargeted cells showed >97% green fluorescent cells indicating the Jump-InTM parental cells are stable up to at least 20
passages
Figure 7: Stability of Jump-InTM retargeted cells
Jump-In™ Parental Cells were retargeted with GFP. Analysis of GFP
expression by flow cytometry at passage 1 (after selection with
blasticidin for 2-3 weeks) and passage 20 showed that at passage 20
the cells were still stably expressing GFP.
NFkB-bla
97.9
pJTI R4 CMV-GFP pJTI R4 CMV-GFP-p53
pJTI R4 CMV-GFP-PRAS40
NFkB-bla (control)GFP-PRAS40GFP-p53GFP
99.0
97.9
0.02
Passage 1
Jump-In™ CHO-K1
Jump-In™ GripTite™ 293
Passage 20
Parental
GFP
Lane 1:
1 Kb Plus Ladder- Lane 2:
parental (negative control)Lane 3:
Jump-In™ CHO- K1 / BamHILane 4:
hygro(R4-vector, positive control 2)
Note:
Image shows different lanes reassembled from the same southern blot
1 2 3 4 Jump-InTM CHO-K1 Southern
Lane 1:
Jump- In™ HEK293 GripTite / BamHI
Lane 2:
Jump- In™ HEK293 GripTite / Hind III Lane 3:
Jump- In™ HEK293 / BamHI (positive
control 1)
Lane 4:
parental (negative control)
Lane 5:
hygro (R4- vector, positive control 2)
Note:
Image shows different lanes
reassembled from the same southern
blot
1 2 3 4 5 Jump-InTM GripTiteTM 293 Southern
Representative Data for Jump- In
Parental Cell Lines
Tel: 800 955 6288 x40266 ● E-mail:
Figure 8: Validation of retargeted cells
Retargeted cells can be validated in a number of different ways, depending on the genetic material used. If there is a fluorescent tag,
flow cytometry can be used to determine the percent of successfully
integrated cells. PCR with primers for the hygromycin resistant gene or
the EF1a-blasticidin resistant gene junction can be used. When
possible, a functional readout can be used, such as the LanthaScreen
p53 cellular assay for detecting phosphorylation at serine 15.
Figure 9: Performance of retargeted cells in CellSensorReporter Gene Assays
Jump-InTM GripTiteTM HEK293 cells were retargeted with either the AP
beta lactamase reporter or the NF-κB beta lactamase reporter. After
antibiotic selection, CellSensor® Assays were run and demonstrated
expected assay windows and EC50 values.
Figure 10: Performance of retargeted cells in LanthaScreen® Assays
Jump-InTM GripTiteTM HEK293 cells were retargeted with GFP
SMAD3, or GFP-PRAS40. After antibiotic selection, Cellular
LanthaScreen® Assays against the indicated modifications were run and
results showed expected assay windows and EC50 values.
Jump-In Griptite HEK293AP-1-bla CellSensor Assay
-4 -3 -2 -1 0 1 2 3 40.51.01.52.02.53.03.54.04.55.05.56.06.5
[PMA], log (nM)
Res
pons
e R
atio
Jump-In GripTiteNF-κκκκB-bla CellSensor assay
-5 -4 -3 -2 -10.51.01.52.02.53.03.54.04.55.05.56.06.5
[TNF-αααα ], log (ng/ml)
Rep
onse
Rat
io
Jump-In
00.00.10.20.30.40.50.60.70.80.91.01.11.2
520
nm /
490
nm
Jump-In HEK293 GripTite GFP-p53 (pS15)
-3 -2 -1 0 1 2 3 4 50.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
[Etoposide], log (nM)
TR
-FR
ET
ratio
Jump-In HEK293 GripTite GFP-SMAD3 (pS423/425)
-2 -1 0 1 2 3 40.040.050.060.070.080.090.100.110.120.130.140.150.160.17
[TGF- ββββ ], log (pM)
TR-F
RE
T r
atio
Jump-InTM Parental Cell Line Kit Application Note Version No. 1
InTM GripTite TM HEK 293 and CHO-K1 Parental Cell Lines
mail: [email protected] ● Web: www.lifetechnologies.com
Retargeted cells can be validated in a number of different ways, depending on the genetic material used. If there is a fluorescent tag,
flow cytometry can be used to determine the percent of successfully
cin resistant gene or
blasticidin resistant gene junction can be used. When
possible, a functional readout can be used, such as the LanthaScreen®
p53 cellular assay for detecting phosphorylation at serine 15.
Performance of retargeted cells in CellSensor®
HEK293 cells were retargeted with either the AP-1
κB beta lactamase reporter. After
Assays were run and demonstrated
Performance of retargeted cells in
HEK293 cells were retargeted with GFP-p53, GFP-
Cellular
Assays against the indicated modifications were run and
values.
Figure 11: Reproducibility of the retargeting
Jump-In™ HEK293 GripTite™ cells were retargeted with GFP in 24
independent transfections (24-well plate). Analysis by flow cytometry
indicated that 100% of wells were retargeted and cell pools were 94.9
98.7% GFP positive.
GripTite HEK293B-bla CellSensor assay
0 1 2 3
], log (ng/ml)
Jump-In HEK293 GripTite GFP-PRAS40 (pT246)
1 2 3 4 5[PI-103], log nM
control
FITC channel
Version No. 1 Page 5 of 6
www.lifetechnologies.com
Reproducibility of the retargeting
were retargeted with GFP in 24
well plate). Analysis by flow cytometry
indicated that 100% of wells were retargeted and cell pools were 94.9 –
FITC channel
Jump-InTM Parental Cell Line Kit Application Note Version No. 1 Page 6 of 6
Representative Data for Jump-In TM GripTite TM HEK 293 and CHO-K1 Parental Cell Lines
Tel: 800 955 6288 x40266 ● E-mail: [email protected] ● Web: www.lifetechnologies.com
Figure 12: GFP expression from retargeting with the control vector
Jump-In™ CHO-K1 cells were imaged following transfection with pJTITM
R4 Exp CMV-GFP-pA. Brightfield and fluorescence microscopy (GFP)
images (10 x objective) were taken at the indicated day of antibiotic
selection with blasticidin. The red circles indicate colonies of blasticidin
resistant retargeted Jump-In™ CHO-K1 cells.
For Research Use Only. Not intended for human or animal therapeutic or diagnostic use. © 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners
