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)
