Description

Step1: Development of a reporter cell line

For in vitro screening assays we are interested in a reporter cell line in a MDA-MB-231 background. The reporter cell line shall stably express a CRE-GFP reporter construct to monitor the activity of cAMP/PKA signal transduction pathways in MDA-MB-231 cells. The Reporter construct is commercially available as plasmid or lentiviral particle e.g from Quiagen: Cignal Lenti CRE Reporter (GFP) Kit: CLS-002G.

The service provider should perform:
1) Stable transduction of cell line with CRE-GFP reporter construct.
2) A Puromycin kill curve of the cell line followed by Puromycin selection of bulk population.
3) Titration curve of Forskolin to proof inducability and range of fluorescent signal.


Step2: Target gene knockdown in established reporter cell line

In addition, this reporter cell line shall stably express either a non-silencing control-shRNA construct or a stable target gene specific-shRNA. Validated target gene shRNA vectors are commercially available as plasmid or lentiviral particle. Selection pressure of the plasmid has to be different from Puromycin e.g. Neomycin.

The service provider should perform:
1) Stable knockout of gene of interest in CRE-GFP reporter cell line and non-silencing ctrl.
2) A Neomycin kill curve of the cell line followed by Neomycin selection of bulk populations.
3) Determine the knockdown efficiency of cell line.
4) Quality control of both cell lines (CRE-GFP-shRNA and CRE-GFP-ctrl) including:
i. Test for contamination with residual virus particles
ii. Test for mycoplasma
iii. Test for Sterility
iv. Test for transgene expression on qRT-PCR level
v. Test for cell viability after thawing
vi. Monitoring of cell growth for at least 72h after thawing

Deliverables:
Several vials of frozen CRE-GFP Reporter cells expressing either target gene shRNA or non-silencing control. Both in above mentioned quality.

Timelines:
• Delivery of final product latest end of February 2015

Purpose:
• Monitor cAMP/PKA signaling pathway activity in cells dependent on gene of interest
• Study chemical compounds or drugs