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Cellular Events Following DNA Transfection

Cellular Events Following DNA Transfection. Expression Transient Expression Stable Expression Day: 0 1-3 3-14. Primary Containment. Three general types of BSC (Class I, II, III)

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Cellular Events Following DNA Transfection

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  1. Cellular Events Following DNA Transfection Expression Transient Expression Stable Expression Day: 0 1-3 3-14

  2. Primary Containment • Three general types of BSC (Class I, II, III) • Class I- not appropriate for handling materials vulnerable to airborne contamination • Class II- inward air flow to protect personnel, HEPA-filtered exhaust for environmental protection. • Class III- Totally enclosed, offers most personnel and environmental protection, for BL3/4 work

  3. Strategies to Improve Each Step of Transfections • Condense DNA • Condensing head group, PEI, spermine, polyarginine • Associate with the cells • Charge (+) recognize (-) charge membrane, Lipid/DNA ratio • Targeting moiety: RGD and integrins, antibodies • Escape endosome • Fusionogenic lipids for example DOPE, help fusion with negatively charge lipids • pH dependant lipids , inc + charge as the pH decrease, favoring fusion • Fusion molecules chloroquine, or peptides • Nuclear entry • Release DNA in cytoplasm • Nuclear entry molecules such as NLS

  4. Preventing Contamination General Practices • Visual/microscopic examination of cultures every time the cells are handled • Grow cultures in antibiotic free media for at least part of the time • Do not share media with other workers or use the same media for different cell types • Inspect media before use

  5. How to Control Growth • Viable contaminants can be neutralized by: • Chemical action - disinfectants • Removal from environment – filter, vacuum, mop, wipe • Thermal • Autoclaving (ovens) – steam, heat and pressure • Depyrogenation (ovens) – dry heat • Radiation – Gamma, E-beam • Filtration

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