corporate
 
 

Simbiosys custom develops stable transfected cell lines of the following types:

Cell lines for recombinant protein production
Cell lines for cell-based assays
Reporter cell lines
   
Starting Material Required (at least one)
Gene accession number
Gene sequence
Target Gene cloned in a cloning / expression vector
   
We will perform the following steps:
Gene Cloning
Insert preparation:
 
PCR amplification of full length ORF of the target gene by reverse transcription from total RNA prepared from specified tissue or cell line. The gene obtained is verified by DNA sequencing
 

Addition of N and / or C termini tags to the target gene by additional PCR amplifications of the target gene with primers encoding for the tags

Mammalian expression vector containing elements for constitutive and high expression of the target protein is prepared by restriction enzyme digestion for cloning the target gene

Cloning of the insert generated into the mammalian expression vector with a KOZAK sequence before ATG start codon

Identification of insert carrying clones
Restriction mapping of insert-carrying clones
Plasmid DNA preparation for transfection into mammalian cell-line
Sequence analysis of cloning junctions
 
Transient Gene Expression
Transient transfection in suitable mammalian cell line by Lipofection
Determination of protein expression by immunofluorescence using protein specific polyclonal or monoclonal antibodies
 
Stable Cell line development
Isolation of single clones by selection and limiting dilution or cloning cylinder
Amplification of the isolated clones
Determination of protein expression by Western blot and immunofluorescence using specific polyclonal or monoclonal antibodies ( if available)
Screening of the clones for identification of the highest expressing cell lines
Isolation of high expressing homogenous stable cell lines and expansion to generate a cell bank
Expression data from 7 passages
   
Deliverable Timeline
Detailed scientific report outlining cloning strategy and references 2 weeks
Data from cDNA cloning and experimental results 4 weeks
cDNA expression clone plasmid preparation (1 g) 2 weeks

Transient transfection of cells expressing target protein and validation of expression using immunofluorescence / western blot

4 weeks

Well characterized cryopreserved and in culture stable cell lines expressing the protein of interest

12 weeks

Whole cell membrane (5 mg/ml) preparation from stable cell line expressing the protein of interest

2 weeks

Transfer of technology to handle cell line and associated assay (immunofluorescence, ELISA, western)

2 weeks

 

 
Assay Development
Cell Isolation & Banking
Cell Line Development
Natural Product Testing
Molecular Biology
Bioinfomatics

 

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