RN46A

Cell Line Origin

Embryonic rat medullary raphe, temperature-sensitive mutant of SV40 large T-antigen, immortalised, serotonergic, neuronal

Cell Line Description

RN46A , an immortalized serotonergic neuronal cell line, was cloned by serial dilution following infection of dissociated embryonic day 13 rat medullary raphe cells with a retrovirus encoding the temperature-sensitive mutant of SV40 large T-antigen (T-ag), RN46A cells are capable of differentiating at 39 °C the non-permissive temperature. Under differentiation conditions, RN46A cells cease dividing, take on a neuronal morphology, and express enhanced levels of NSE and all three NF proteins. Differentiated RN46A cells express low-affinity nerve growth factor (NGF) receptor (p75NGFR) and are i mMunoreactive using an antibody that recognizes the carboxy-terminal 13 amino acids of all three trk proteins (pan-trk). Both i mMunoreactivities could be potentiated by treatment with brain-derived neurotrophic factor (BDNF), NGF, and adrenocorticotropic hormone, fragment 4-10 (ACTH4-10). Differentiated RN46A cells express low levels of tryptophan hydroxylase (TPH) i mMunoreactivity, which could be enhanced by treatment with ACTH4-10, BDNF, or NGF. Low levels of serotonin i mMunoreactivity are detected in differentiated RN46A cells, and this was potentiated by differentiating RN46A cells with BDNF for 8 d and 40 mM KCl for days 4-8. RN46A cells should prove useful to elucidate intracellular mechanisms that control neurofilament assembly and 5-HT expression in differentiating raphe neurons.

DNA Profile

Not specified

Culture Medium

DMEM:F12 (1:1) (D8062) + L-Glutamine (G7513) + 10% FBS / FCS (F2442) + 0.25mg/ml Geneticin (G418). Alternatively CNS medium can be used (see Kawamoto & Barrett 1986). Differentiation can be induced as follows: cells growing at 33 °C are sub-cultured onto collagen/fibronectin matrix (100 μg/cm² air-dried collagen I from rat tail followed by 1 μg/cm² fibronectin). Sub-confluent cells (75%) are shifted to 39 °C. The culture medium is changed to DMEM:F12 (1:1) (D8062) + 1%(w/v) bovine serum albumin (BSA) + 1 μg/ml bovine transferrin + 5 μg/ml bovine insulin + 100 nM putrescine + 20 nM progesterone.

Subculture Routine

Split subconfluent cultures (70-80%) 1:2 to 1:5 using 0.25% trypsin/EDTA; 5% CO₂; 33 °C. Suggested seeding density 2-4 x 10,000 cells/cm². Doubling time 19hrs.

Other Notes

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