Customized Services

For Stable Knock-Down or Over-Expression Cell Line Generation

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.

Method

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-expression or knock-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 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.

 

Vector Set Selection Marker Approach for stable Cell Line generation Application

pLKO.1

(Constitutive) 		Puromycin 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
Neomycin Drug selection Knockdown of multiple targets by miR-shRNAs
Hygromycin 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

pSLIK system

pSLIK system: inducible lentiviral system (Tet-On)

pLKO.1 system

pLKO.1 system: constitutive lentiviral system

 

Workflow (using the lentiviral 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.

Pricing and Timeline

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.

Services
Price (in USD)
Time line
Probe design, construction and validation
$1,330 /construct
5-7 weeks
pSLIK system
Polyclonal cell line generation and validation
$6,380 /cell line
12-15 weeks
Monoclonal cell line generation and validation
$9,460/cell line
18-23 weeks
pLKO.1
Polyclonal cell line generation and validation
$5,580 /cell line
7-9 weeks
Monoclonal cell line generation and validation
$8,660/cell line
13-17 weeks

Examples

miR-shRNA

Example 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.

miR-shRNA

Example 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.

 

 

Contact Info:

Ms. Wei Zhang
Project Management
Office: +86-21-51319016
           +86-21-50802786 Ext: 188
Fax:    +86-21-50802783
Email: service@shanghaigenomics.com
Service Web: www.genomicservice.com

 

 

Shanghai Genomics, Inc.

www.shanghaigenomics.com

647 Song Tao Road, Building 1,Zhangjiang Hi-Tech Park

Shanghai 201203,P.R.China