Stable Transfection Protocols

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. - [Read Antisense Oligonucleotide Transfection of RAW 264.7 Cells with FuGENE 6 in a 24-Well Dish]

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. - [Read DNA Transfection by Electroporation]

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. - [Read Establishment of Stable Transfectant of CHO Lec Cells Protocol]

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. - [Read Tetracycline as Regulator of Inducible Gene Expression III]

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. - [Read Tetracycline as Regulator of Inducible Gene Expression Protocol]

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. - [Read Tetracycline as Regulator of Inducible Gene Expression Protocol II]