UiPath Solutions Management Preview - Northern CA Chapter - March 22.pdf
Customized stable cell line services
1. CustomizedCustomizedCustomizedCustomized ServicesServicesServicesServices
ForForForFor StableStableStableStable Knock-DownKnock-DownKnock-DownKnock-Down orororor Over-ExpressionOver-ExpressionOver-ExpressionOver-Expression
CellCellCellCell LineLineLineLine GenerationGenerationGenerationGeneration
Stable cell lines with specific gene over-expression or knock-down are very
helpful in gene function analysis, target discovery, target validation, assay
development, and compound screening. However, generation of stable cell
lines is a time-consuming and expensive process. In the last 4 years,
Shanghai Genomics has established a team of experts for stable cell line
generation. We have successfully generated hundreds of stable cell lines to
meet the need of our customers.
MethodMethodMethodMethod
Shanghai Genomics typically uses non-viral (liposome) and viral (lentivirus)
platforms for stable cell line generation. For some cell lines, like HEK293
and HeLa, the liposome method can achieve high transfection efficiency.
Compared to liposomal or other chemical vehicles, a lentiviral approach has
three distinct advantages:
1. The pseudotype virus has a broad range of host cell lines and is able to
infect both dividing and non-dividing cells, making it especially suitable for
some differentiated cell lines such as neurons, macrophages, hematopoietic
stem cells, retinal photoreceptors and muscle and liver cells.
2. The virally encoded integrase greatly facilitates exogenous gene
integration that ensures long-term and stable expression.
3. The pseudo-type lentivirus is engineered so that it does not replicate and
is safe for most lab operations. Lentiviral platform is most frequently chosen
by customers. Other retrovirus system is also available. Details can be
provided if this platform is applicable to customer's cell line of interest.
Over-expressionOver-expressionOver-expressionOver-expression orororor knock-downknock-downknock-downknock-down
Over-expression or knock-down is one of the most widely used tools for
gene function research and disease target validation. Shanghai Genomics has
2. successfully generated hundreds of stable cell lines with either gene
over-expression or knock-down.
1. Over-expression cell lines: Many mammalian expression vectors or
lentiviral vectors are suitable for this purpose. Stable cell lines have been
generated in-house with pGL4; pcDNA3.1; pEF_Bos; pLenti-DEST vectors.
We may help customers to select a proper vector for their customized cell
line generation.
2. Knock-down cell lines: RNAi techniques are widely used for gene
knockdown applications. shRNA and miR-shRNA (a pseudo-type miRNA)
are major techniques used to generate SG's knockdown cell lines. Since
long-term suppression using pol III shRNAs can be problematic especially
for studies of genes that are critical to cell proliferation and differentiation,
we provide not only constitutive but also conditional knock-down. The table
below lists two major sets of lentiviral RNAi vectors used by SG for
knockdown cell line generation.
VectorVectorVectorVector
SetSetSetSet
SelectionSelectionSelectionSelection
MarkerMarkerMarkerMarker
ApproachApproachApproachApproach forforforfor
stablestablestablestable CellCellCellCell LineLineLineLine
generationgenerationgenerationgeneration
ApplicationApplicationApplicationApplication
pLKO.1
(Constitu
tive)
Puromyci
n
Drug selection
Knockdown of the target by
shRNA
EGFP Sort by FACS
Knockdown of the target by
shRNA
pSLIK
(Tet On)
Zeocin Drug selection
Knockdown of multiple targets
by miR-shRNAs
Neomyci
n
Drug selection
Knockdown of multiple targets
by miR-shRNAs
Hygromy
cin
Drug selection
Knockdown of multiple targets
by miR-shRNAs
CD4 Drug selection
Knockdown of multiple targets
by miR-shRNAs
Venus Sort by FACS
Knockdown of multiple targets
by miR-shRNAs
3. pSLIKpSLIKpSLIKpSLIK system:system:system:system: inducibleinducibleinducibleinducible lentivirallentivirallentivirallentiviral systemsystemsystemsystem (Tet-On)(Tet-On)(Tet-On)(Tet-On)
pLKO.1pLKO.1pLKO.1pLKO.1 system:system:system:system: constitutiveconstitutiveconstitutiveconstitutive lentivirallentivirallentivirallentiviral systemsystemsystemsystem
WorkflowWorkflowWorkflowWorkflow (using(using(using(using thethethethe lentivirallentivirallentivirallentiviral system)system)system)system)
There are many mature non-viral transfection products and many optimized
protocols available for stable cell line generation on the market. The
following flowchart shows the general workflow with the lentiviral infection
system. Generally, target expression will be investigated in established
stable cell lines. Mycoplasma testing and STR profiling are also done as part
of regular quality control to ensure mycoplasma free and identity between
master and established stable cell lines.
4. PricingPricingPricingPricing andandandand TimelineTimelineTimelineTimeline
The table below shows a rough price and timeline for customized stable cell
line generation using lentiviral infection. For different targets and cell lines,
or using nonviral transfection approach, please contact us for prices and
timelines.
ServicesServicesServicesServices PricePricePricePrice (in(in(in(in USD)USD)USD)USD) TimeTimeTimeTime linelinelineline
Probe design, construction
and validation
inquiry 5-7 weeks
pSLI
K
syste
m
Polyclonal cell line
generation and
validation
inquiry 9-10 weeks
Monoclonal cell
line generation and
validation
inquiry 13-14 weeks
pLK
O.1
Polyclonal cell line
generation and
validation
inquiry 8-9 weeks
Monoclonal cell
line generation and
validation
inquiry 12-13 weeks
ExamplesExamplesExamplesExamples
5. ExampleExampleExampleExample 1:1:1:1: miR-shRNA against luciferase was introduced into the
293T-Luc cell line by lentiviral infection and then sorted by FACS.
Constitutively expressed Luciferase was knocked down in the presence of
DOX.
ExampleExampleExampleExample 2:2:2:2: miR-shRNA against gene 1 or over-expression of gene 2 was
introduced into the master cell line by lentiviral infection and drug selection.
Significant knockdown or over-expression was observed by both RT-QPCR
and Western blot in the presence of DOX.
ContactContactContactContact Info:Info:Info:Info:
Linna Green
Project Management
Tel: 1-631-626-9181
Fax: 1-631-614-7828
Email: info@creative-biogene.com
(Creative(Creative(Creative(Creative DynamicsDynamicsDynamicsDynamics andandandand CDCDCDCD Bocsci)CDBocsci)CDBocsci)CDBocsci)CD,,,, Inc.Inc.Inc.Inc.
www.creative-biogene.com
45-16 Ramsey Road Shirley, NY,11967,USA