Stable Transfection Protocols

Antisense Oligonucleotide Transfection of RAW 264.7 Cells with FuGENE 6 in a 24-Well Dish The AfCS is utilizing antisense technology to manipulate signaling protein expression in the RAW 264.7 macrophage-like cell line. This can be achieved by the transfection of gene-specific antisense oligonucleotides (ASOs). The following procedure involves the transfection of ASOs into RAW 264.7 cells using FuGENE 6 transfection reagent. Subsequently, the isolated total RNA or protein from these transfected cells can be used to assess the level of mRNA or protein knockdown, respectively.

DNA Transfection by Electroporation Pulsed electrical fields can be used to introduce DNA into a wide variety of animal cells. Electroporation works well with cell lines that are refractive to other techniques, such as calcium phosphate-DNA coprecipitation. But, as with other transfection methods, the optimal conditions for electroporating DNA into untested cell lines must be determined experimentally.

Establishment of Stable Transfectant of CHO Lec Cells Protocol CHO Lec3.2.8.1 cells have 4 independent mutations in the N and O glycosylation pathways. When cultured with alpha-glucosidase I inhibitor N-butyl-deoxynojirimycin, glycoproteins produced in CHO Lec3.2.8.1 cells are completely susceptible to Endo H digestion. Endo H cleaves chitobiose, leaving a single N-linked N-acetylglucosamine per site which is ideal for maintenance of protein solubility and special carb-protein interactions, such as between the first N-acetyl glucosamine residue and tryp.

Tetracycline as Regulator of Inducible Gene Expression III Stably transfected cells, generated in the first two stages of the procedure, are induced for expression of the target gene. After harvesting and lysis, the lysates are analyzed by SDS-PAGE and immunoblotting.

Tetracycline as Regulator of Inducible Gene Expression Protocol Protocol uses an autoregulatory system in which the transcriptional trans-activator tTA drives its own expression and that of a target gene. The first stage of the procedure describes how to generate stable lines of NIH-3T3 cells that express either tTA alone or tTA and the tetracycline-regulated target gene.

Tetracycline as Regulator of Inducible Gene Expression Protocol II This stage of the procedure describes the transfection with target genes of cell lines already expressing inducible tTA. In this example, the target genes are transfected on a plasmid that carries puromycin resistance as a selectable marker.