Sequential transfection

Sequential Transfection of Plasmid DNA in a T-75 Flask and siRNA in 24-Well Plates (for Adherent Cells)... 

Sequential Transfection. A simple approach to boosting expression is to repeat the transfection on previously transfected cells but with selectable markers not used in the first transfection. Xoma (Berkeley, CA), Sunol Molecular (Miramar, FL), and ICOS (Bothell, WA) have used this approach successfully. Fivefold or greater improvements in expression can be achieved in a single sequential transfection. ICOS reports the added advantage of balancing heavy and light chain ratios to improve the secretion and expression of recombinant monoclonal antibodies. 

Sequential hydroformylation/reductive amination of dendritic perallylated polyglycerols with various amines in a one-pot procedure to give dendritic polyamines in high yields (73-99%). Furthermore, the use of protected amines provides reactive core-shell-type architectures after deprotection. These soluble but membrane filterable multifunctional dendritic polyamines are of high interest as reagents in synthesis or as supports in homogeneous catalysis as well as nonviral vectors for DNA-transfection (Scheme 18) [65]. 

Fig. 14. Detection of -galactosidase activity in cells using F NMR. Sequential F NMR spectra of LNCaP C4-2 prostate cancer cells transiently transfected with lacZ (1.0 x 10 ) in phosphate buffered saline (PBS) (0.1 M, pH = 7.4, 700 ml) at 37 C following addition of GFPOL (1.84 mg, 5.27 mmol). F NMR spectra were acquired in 102 s each, and enhanced with an exponential line broadening 40 Hz. In each spectrum, GFPOL occurs on the left with liberated FPOL aglycon appearing at right ( ).

In most cases, cells are grown in a serum-containing environment during the early stages of cell line development, such as transfection and selection. Once a SF medium with a good nutrient balance is chosen, the next step is to adapt cells to SF growth. Two adaptation strategies, sequential adaptation and starve/save adaptation, are described. 

To introduce two vectors carrying different genes into the same CEF cells, separate transfections (using vectors containing different subgroup env genes) should be done sequentially. The CEF/DFl cells should be passaged two or three times between transfections. 

Fig. 2. Image acquisition and processing steps to determine the transport of ts-045-G to the plasma membrane. HeLa cells were transfected with siRNAs on LabTek arrays as they are described in Chapter 1 of this issne. The ts-045-G transport assay was carried out as described in protocol 1.1 as described earlier in this chapter. Images were acquired sequentially using a lOX objective on a Scan R system using filters to detect specifically DAPI stained nuclei (A), Cy3 stained ts-045-G at the plasma membrane (B), and CFP-tagged ts-045-G (C). Images DT were generated as described in protocol 1.2. earlier in this chapter. R in (G) is the ratio of ts-045-G at the plasma membrane (measured in H) to ts-045-G expressed in cells (measured in I). Resnlts for siRNAs targeting the COPI component /3-COP, the COPII component Sec31p, and a p24 related membrane protein p26 are shown. The valnes are the average of two independent experiments (Bar = 50 /tm).

Tan Y, Liu F, Li Z, Li S, Huang L. Sequential injection of cationic liposome and plasmid DNA effectively transfects the lung with minimal inflammatory toxicity. Mol Ther 2001 3(5) 673-682. 

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