How do you establish a stable transfection cell line?
Ensure that only one cell is present per well after the transfer.
- Step 1 : Transfect cells. Transfect the cells using the desired transfection method.
- Step 2 : Passage cells with antibiotic.
- Step 3 : Monitor for cell “islands”
- Step 4 : Isolate colonies.
- Step 5 : Transfer single cells.
What does mean stable cell line?
Generation of a stable cell line refers to the process of developing homogenous populations of cells that demonstrate expression of a transfected gene insert. The transfected gene integrates into the genome of the host cell, and as a result, are able to express the transfected genetic material.
What is a transfected cell line?
Broadly defined, transfection is the process of artificially introducing nucleic acids (DNA or RNA) into cells, utilizing means other than viral infection.
How long does it take to make a stable cell line?
Stable production of monoclonal antibodies in CHO cells is still the method of choice for the production of most biopharmaceuticals. However, generating stable cell lines is a laborious and time-consuming process starting at 4-6 months.
How long does it take to generate stable cell line?
Timeline for Stable Cell Line Generation. Stable cell line generation can take a total of 9-12 weeks to establish the cell line. It is ideal to first determine the optimal antibiotic concentration for selection for each cell type; this can take up to 1 week.
How long do transfections last?
24 to 96 hours
Depending on the construct used, transiently expressed transgene can generally be detected for 1 to 7 days, but transiently transfected cells are typically harvested 24 to 96 hours post-transfection. Analysis of gene products may require isolation of RNA or protein for enzymatic activity assays or immunoassays.
How do you maintain a stable cell line?
Stable cell line generation protocol
- Generate a kill curve to determine the optimal selection antibiotic concentration.
- Transfect cells with desired plasmid construct(s)
- Select and expand stable polyclonal colonies.
- Identify single clones by limited dilution and expansion.
- Transfer clones and assess expression.
What can you do with transfected cells?
The main purpose of transfection is to study the function of genes or gene products, by enhancing or inhibiting specific gene expression in cells, and to produce recombinant proteins in mammalian cells [3].
How much puromycin should I take for selection?
In mammalian cells, the recommended working concentration range for puromycin is 0.5 – 10 µg/ml. Different cell types and cell culture conditions may require different concentrations of selection antibiotic.
What is the function of the pegfp N1 promoter?
A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pEGFP-N1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
How can I Digest a vector with pegfp-n1?
If you wish to digest the vector with this enzyme, you will need to transform the vector into a dam– and make fresh DNA. Description: pEGFP-N1 encodes a red-shifted variant of wild-type GFP (1–3) which has been optimized for brighter fluorescence and higher expression in mammalian cells.
Can eGFP be transfected into mammalian cells?
The recombinant EGFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (7). pEGFP-N1 can also be used simply to express EGFP in a cell line of interest (e.g., as a transfection marker).
What is the N-terminus of pegfp-n1?
pEGFP-N1 is between the immediate early promoter of CMV and the EGFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of EGFP if they are in the same reading frame as EGFP and there are no intervening stop codons.