Transfection of mammalian cells

Learn that transfection is the process of inserting DNA or RNA into eukaryotic cells, with DNA targeted to the nucleus and RNA potentially delivered to the cytoplasm. Understand the distinction between transfection for DNA versus RNA molecules like siRNA and mRNA.

Examine the three major categories of transfection: chemical methods (calcium phosphate, DEAE-dextran), lipid-based methods (liposome), and physical methods (gene gun, electroporation). Compare properties, timelines, and applicability of each method for different cell types.

Understand that transfection enables studying gene function by modulating gene expression, blocking or turning on protein synthesis, and transferring DNA into embryonic stem cells for development studies. The goal is to manipulate cellular genetics while keeping cells viable.

Learn how positively charged calcium ions bind to the negatively charged DNA phosphate backbone, forming calcium phosphate precipitate beads that settle on cells. The cells then take up the intact DNA strands through endocellular mechanisms.

Understand that liposomes are phospholipid vesicles with hydrophilic interiors that encapsulate negatively charged DNA with positively charged lipids to form lipoplexes. These lipoplexes interact with the negatively charged cell membrane, merging with it or being engulfed by endocytosis for DNA delivery to the nucleus.

Recognize that lipoplexes can either fuse with lysosomes leading to DNA degradation, or escape endosomes to allow free DNA to enter the nucleus through nuclear pores. Cell culture conditions can be optimized to prevent lysosomal fusion when intact DNA insertion is desired.

Learn that electroporation uses high-voltage pulses to create temporary pores in the cell membrane, allowing nucleic acids to pass through. The pores remain open for only a fraction of a second before resealing.