Generation Of Stable Cell Lines Protocol

Protocol of stable cell line generation.

Generation of stable cell lines protocol.

Stable expression of target gene in cell lines overcomes the low transfection efficiency. Lentivirus is really an efficient way to generate stable cell lines. So far generation of stable cell lines has been a major challenge for many cell types e g jurkat mcf7 or u937 since overall transfection efficiencies and or integration frequencies have been low. Two cell lines created using g ie n tr tata that met the optimal criteria were tested for de repressive and inducible properties using a combination of dox and vp16.

Fig 5 diagrams the generation of cell lines using hsv ie bidirectional promoter system showing how expression is controlled using doxycycline and vp16. The basic steps in the generation of stable cell lines expressing chemokine receptors are as follows. The transfected gene integrates into the genome of the host cell and as a result are able to express the transfected genetic material. Protocol for generation of stable cell lines 2.

Generate a kill curve to determine the optimal selection antibiotic concentration. Protocol of stable cell line generation 1. The protocol for generating stable cell lines requires several steps as shown below. Lentiviruses are used very widely to generate stable expression mammalian cell lines.

Integration into host cell genome it mediates long term and stable expression of exogenous genes with a simple system for. It is applied to the production of recombinant proteins gene function studies as well as drug discovery assays. They are used for both gene down regulation by using shrna or for gene up regulation by using orf of gene of interest. Transfect cells with desired plasmid construct s select and expand stable polyclonal colonies.

While common transfection methods such as lipofection can be used for the stable expression in easy to transfect cell lines e g hela cos 7. This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector. 1 applications types major challenges transfection methods selection marker methods and culture conditions of stably transfected cell lines. The generation of stably transfected cell lines is critical for a wide range of applications.

Stable cell line generation protocol. Unlike the short term protein expression observed using transient transfection approaches generating cell lines using lentiviral vectors enables long term protein expression studies. Generation of a stable cell line refers to the process of developing homogenous populations of cells that demonstrate expression of a transfected gene insert.