RETROVIRAL-MEDIATED FUNCTIONAL ANALYSIS OF CELL CYCLE CONTROL PROTEINS Sample Clauses

RETROVIRAL-MEDIATED FUNCTIONAL ANALYSIS OF CELL CYCLE CONTROL PROTEINS. Cell cycle control genes selected by Xxxxxxx will be transferred into Rigel's retroviral system for stable expression and functional analysis in selected tumor cell lines. Encoding sequences will be cloned into the multiple cloning region of the basic retroviral vector construct (Appendix H). An internal ribosome entry site placed immediately 3' of the multiple cloning site, drives cap-independent translation of downstream encoding sequences. This allows co-translational selection with a downstream FACS-selectable marker (e.g. GFP). Infectious retroviral particles are produced by transient transfection of retroviral vector, constructs into high efficiency packaging cell lines (Appendix G), harvested, and used to infect target cell lines for stable integration into the target cell genome. Optimal infection protocols will be developed for each cell line using- a GFP control vector. A generic protocol is detailed in Appendix G. Examples of infection rate for various tumor cell lines is shown in Table 2. TABLE 2. INFECTION RATES WITH RIGEL RETROVIRAL CONSTRUCTS FOR VARIOUS TUMOR CELL LINES. --------------------------------------------------------------------------------------------------------------------- CELL LINE INFECTION EFFICIENCY (%) --------------------------------------------------------------------------------------------------------------------- A549 (non-small cell lung cancer) >80 HeLa (cervical carcinoma) >70 T47D (breast cancer) >70 SW480 (colorectal cancer) >70 CEM (T-lymphoblastoid leukemia) >30 --------------------------------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------- In most cases, transduction rates of >70% are achievable with limited optimization of the standard protocol. Occasionally, cells express low levels of the retroviral receptor recognized by the retroviral envelope protein. This may be compensated by engineering the target cell lines to express higher receptor levels by introducing die ecotropic envelope protein receptor (EcoR), a basic amino acid transporter. For example, infection efficiency of Jurkat T-lymphoblastoid leukemia cells was enhanced from ~15% to >90% in EcoR-expressing lines. Alternatively, retroviral vector particles may be pseudotyped with the Vesticular Stomatitis Virus G-protein (VSV-G), which interacts with membrane lipids, (phosphatidylserine) to promo...
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