Efficient DNA-mediated transfer of selectable genes and unselected sequences into differentiated and undifferentiated mouse melanoma clones

Abstract

We have found that three phenotypically dissimilar mouse B16 melanoma subclones are competent recipients for DNA-mediated gene transfer. Two of these approach and a third, amelanotic clone B78H1, surpasses mouse LTK cells in frequencies of transferent colony formation after treatment with either of two codominantly selectable plasmid vectors, pSV2gpt or pGCcos3neo. Melanoma transferents incorporate both selectable plasmid-homologous sequences and substantial amounts of unselected donor DNA into their cellular DNAs. In addition they retain the distinctive states of differentiation characteristic of the untreated clones. Frequencies of pGCcos3neo-mediated transfer of neo gene-encoded antibiotic resistance into B78H1 can reach 10(-2) in response to treatment with as little as 15 ng plasmid/ml coprecipitate/dish. B78H1 cells readily give rise to "secondary" transferents for the neo gene after treatment with DNA from a "primary" B78H1 neo transferent. This gene transfer system has potential applications for study of regulation of melanoma and neural crest differentiation and malignancy.

Anti-tumor immunity induced by murine melanoma cells transduced with the Mycobacterium tuberculosis gene encoding the 38-kDa antigen

“…9 The supernatant of the producer cell lines was tested for viral titer and antigen expression by FACScan analysis with MAb HBT12 and used for infection of B16-B78 murine melanoma cells. 11 Transduced cells expressed the Mycobacterium tuberculosis Ag38 transcript and produced the bacterial protein as detected by RT-PCR ( Figure 2a) and Western blot (Figure 2b), respectively. Reactivity with MAb HBT12 revealed two bands in the total cell membrane extract at 47 kDa and 42 kDa, corresponding, respectively, to the unprocessed form, which contains both the leader and transmembrane sequences, and to the mature protein after cleavage of the leader sequence.…”

“…Mycobacterium gene transduction and expression in cells B16-B78 murine melanoma cells 11 (6 × 10 5 ) were cultured for 2 h with 5 ml of undiluted viral supernatant from the packaging cell line and 8 g/ml of polybrene; fresh medium was then added to dilute the supernatant (1 : 2) for the following 24 h. To improve infection efficiency, the same infection cycle was repeated five times. Individual colonies as well as bulk cultures of G418-selected (1 mg/ml) melanoma cells were tested for 38-kDa antigen gene expression by RT-PCR, FACScan analysis and Western blotting.…”

Adeno-associated virus gene expression inhibits cellular transformation by heterologous genes.

“…ptk (HSV thymidine kinase gene) (45), pXBAC (E4 gene) (16), pSV2cat (11), and pMMTneo and pBPV/ MMTneo (28) plasmids were generous gifts of M. Chao, R. J. Samulski, E. Rifkin, and P. Howley, respectively. The neo vector, pGCcos3neo, has been described previously (12) and was derived from pSV2neo (40), differing mainly by the insertion of a lambda cos sequence outside of the neo gene.…”

Decreased adhesion to endothelial cells and matrix proteins of H-2Kb gene transfected tumour cells

“…Tumour cells B78H1 is an amelanotic clone originally obtained in the laboratory of S. Silagi from B16 melanoma (Graf et al, 1984); it shows no H-2 expression even after interferon-'y treatment. The derivation of control (transfected with pSV2neo gene encoding resistance to the neomycin analogue G418) and H-2Kb transfectants has been described previously (De Giovanni et al, 1991).…”