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UK Health Security Agency

RN46A

RN46A

Catalogue No.

12061302

Cell Line Name

RN46A

Cell Line Description

RN46A , an immortalised 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 immunoreactive using an antibody that recognizes the carboxy-terminal 13 amino acids of all three trk proteins (pan-trk). Both immunoreactivities 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) immunoreactivity, which could be enhanced by treatment with ACTH4-10, BDNF, or NGF. Low levels of serotonin immunoreactivity 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.

Characteristics

Products

Undifferentiated RN46A cells express low levels of neuron-specific enolase (NSE) and low (NF-L)-and medium (NF-M)- but not high (NF-H)-molecular-weight neurofilament proteins.

Tissue of Origin

Embryonic rat (day 13) medullary raphe

Morphology

Fibroblast morphology while proliferating and neuronal on differentiation

Disease

None Stated

Culture Conditions

Cell Type

Fibroblast morphology while proliferating and neuronal on differentiation

Subculture Routine

Split subconfluent cultures (70-80%) 1:2 to 1:5 using 0.05% trypsin/EDTA; 5% CO₂; 33°C. Suggested seeding density 2-4x10,000 cells/cm². Doubling time 19hrs.

Culture Medium

DMEM: Ham's F12 (1:1) + 2mM Glutamine + 10% Foetal Bovine Serum (FBS) + 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: Ham's F12 (1:1) + 1%(w/v) bovine serum albumin (BSA) + 1µg/ml bovine transferrin + 5µg/ml bovine insulin + 100nM putrescine + 20nM progesterone.

Growth Mode

Adherent

Additional Info

Depositor

Dr Scott R Whittemore, Kentucky Spinal Cord Injury Research Centre, 511 S. Floyd St., Rm 616A, University of Louisville School of Medicine, Louisville, KY 40202. USA

Country of Origin

United States

GMO Status

Genetically Modified Organism Class 1 (GMO1)

Hazard Group (ACDP)

Hazard Group (ACDP) 2

Applications

References

White LA, Eaton MJ, Castro MC, Klose KJ, Globus MY, Shaw G, Whittemore SR. 1994 Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons. J Neurosci. 14:6744-53.PMID: 7965075.

Bibliography

Eaton MJ, Whittemore SR. 1996 Autocrine BDNF secretion enhances the survival and serotonergic differentiation of raphe neuronal precursor cells grafted into the adult rat CNS. Exp Neurol. 140:105-14.PMID: 8690054. Kawamoto JC, Barrett JN.1986 Cryopreservation of primary neurons for tissue culture. Brain Res. 384:84-93.PMID: 3791002.

Available Formats

  • Frozen

If use of this culture results in a scientific publication, it should be cited in the publication as: RN46A (ECACC 12061302).

Unless specified otherwise, at ECACC we routinely handle all of our cell lines at containment level 2 in accordance with the ACDP guidelines (Advisory Committee on Dangerous Pathogens) (UK). All cell cultures have the potential to carry as yet unidentified adventitious agents. It is the responsibility of the end user to ensure that their facilities comply with biosafety regulations for their own country. ACDP Guidance: Biological agents: Managing the risks in laboratories and healthcare premises.

The Culture Collections represent deposits of cultures from world-wide sources. While every effort is made to ensure details distributed by Culture Collections are accurate, Culture Collections cannot be held responsible for any inaccuracies in the data supplied. References where quoted are mainly attributed to the establishment of the cell culture and not for any specific property of the cell line, therefore further references should be obtained regarding cell culture characteristics. Passage numbers where given act only as a guide and Culture Collections does not guarantee the passage number stated will be the passage number received by the customer.

Cultures supplied by Culture Collections are for research purposes only. Enquiries regarding the commercial use of a cell line are referred to the depositor of the cell line. Some cell lines have additional special release conditions such as the requirement for a material transfer agreement to be completed by the potential recipient prior to the supply of the cell line. Please view the Terms & Conditions of Supply for more information.