Stable transfectants

Peptides with (K)ig domains can also form stable transfection particles with large DNA molecules including PI artificial chromosomes of 110 kb (28) and bacterial artificial chromosome (BAC) DNA constructs in a range of sizes up to 250 kb (29). The size, determined by atomic forces microscopy, of lipopolyplex particles formed with BAC DNA is directly proportional to the size of the BAC and transfections performed with equimolar amounts of 100-kb BACs and 8-kb pDNA are similar in efficiency. 

U2-OS cells stable transfected with SSTR2/(3-arrestin2 YFP (see Note 1). 

Transform into E. coli cells, prepare minipreps and identify correct clones by digesting with Sail and Hindlll. Correctly ligated clones show two bands of 1.6 and 3.0 kb, plasmids without insert show only the 4.3 kb vector band. M13 forward, Ml3 reverse, and bpA-for primers can be used for sequence confirmation. Prepare Maxipreps and use two independent plasmids of pRMCE-U6-shRNA for stable transfection by RMCE in ES cells. 

Use the correspondent constructs in the conditional state, which still contain the floxed stop cassette for ligation into RMCE vectors and subsequent stable transfection into ES cells by RMCE. 

Sbicego, S., Schnaufer, A. and Blum, B. (1998) Transient and stable transfection of Leishmania by particle bombardment. Molecular and Biochemical Parasitology 94, 123-126. 

DEAE-dextran. Like the calcium phosphate co-precipitation method, the DEAE-dextran technique was originally developed to increase the viral infectivity of animal cells, and its application was later extended to transfection processes. Although it is simple, efficient, and appropriate for transient expression, its use for stable transfections has not given satisfactory results. The transfection efficiency of this method can be increased by treating cells with glycerol or DMSO. The DNA is incorporated by endocytosis, and thus exposed to extreme pH levels and cellular nucleases, which may explain, to a certain extent, the high frequency of mutations observed when transfecting by this method (Calos et al., 1983). This transfection technique can be applied to both adherent and suspension cell lines. For detailed transfection protocols, the works by Keown et al. (1990) and Kaufman (1997, 2000) are recommended. 

MDC biological activity is assayed by calcium flux or chemotaxis using either primary cells or more commonly stable transfectants expressing CCR4 (Imai et al, 1998). 

Cell and tissue implants Cell suspensions are obtained by trypsinization of confluent cell monolayers. Five microliters containing 2 X 10 cells in medium supplemented with 10% serum are introduced in the corneal micropocket. When the over expression of growth factors by stable transfection of a specific cDNA is studied, one eye is implanted with transfected cells and the other with the wild type cell line. A 0ien tissue samples are tested, samples of 2-3 mg are obtained by cutting the original fragments under sterile conditions. The angiogenic activity of tumor samples is compared with macroscopically healthy tissue. 

---