RN33B (ATCC® CRL-2825)

Organism: Rattus norvegicus, rat  /  Cell Type: neuronalSV40 large T antigen transfected  / 

Organism Rattus norvegicus, rat
Cell Type neuronalSV40 large T antigen transfected
Product Format frozen
Morphology spindle-shaped at 33C; neurite-like at 37C
Culture Properties adherent
Biosafety Level 2 cells containing SV40 viral DNA sequences
Age 12.5 days gestation embryo
Strain Sprague-Dawley
Applications
The cells can be used as a model of neuronal differentiation in vitro.
Immunohistochemical and Northern blot analysis revealed high levels of low affinity NGF receptor protein and mRNA in differentiated RN33B cells.
At the non-permissive temperature of 37C to 39C SV40 T antigen expression is markedly decreased and RN33B cells cease mitotic activity and differentiate with phase bright cell bodies and 'neuritic-like' processes.
RN33B is an neuronal cell line derived from medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen.
At the permissive temperature of 33C, RN33B cells divide and express SV40 T antigen, vimentin, nestin, diffuse neuron-specific enolase, and low and medium molecular weight neurofilament immunoreactivities.
PCR analysis demonstrated the presence of trkB, but not trkA or trkC, mRNA in both undifferentiated and differentiated RN33B cells.
Storage Conditions liquid nitrogen vapor phase
Images
Derivation
RN33B is an neuronal cell line derived from medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen. The cells were cloned by serial dilution. At the permissive temperature of 33C, RN33B cells divide and express SV40 T antigen, vimentin, nestin, diffuse neuron-specific enolase, and low and medium molecular weight neurofilament immunoreactivities. At the non-permissive temperature of 37C to 39C SV40 T antigen expression is markedly decreased and RN33B cells cease mitotic activity and differentiate with phase bright cell bodies and 'neuritic-like' processes. Differentiated RN33B cells express enhanced neuronal-specific protein expression but do not synthesize astrocytic or oligodendrocytic-specific proteins. Moreover, differentiated RN33B cells returned to 33C re-express T antigen, but do not de-differentiate or begin dividing. Immunohistochemical and Northern blot analysis revealed high levels of low affinity NGF receptor protein and mRNA in differentiated RN33B cells. PCR analysis demonstrated the presence of trkB, but not trkA or trkC, mRNA in both undifferentiated and differentiated RN33B cells. The cells can be used as a model of neuronal differentiation in vitro.
Receptor Expression
nerve growth factor (NGF), expressed
Genes Expressed
enolase,nestin,neurofilament,vimentin
Cellular Products
enolase
nestin
neurofilament
vimentin
Effects
Yes, differentiates when transplanted to adult rat subretina
No, do not form colonies in soft agar
Yes, migrates when transplanted to adult and neonatal rat brain
Comments
RN33B is an neuronal cell line derived from medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen. The cells were cloned by serial dilution. At the permissive temperature of 33C, RN33B cells divide and express SV40 T antigen, vimentin, nestin, diffuse neuron-specific enolase, and low and medium molecular weight neurofilament immunoreactivities. At the non-permissive temperature of 37C to 39C SV40 T antigen expression is markedly decreased and RN33B cells cease mitotic activity and differentiate with phase bright cell bodies and 'neuritic-like' processes. Differentiated RN33B cells express enhanced neuronal-specific protein expression but do not synthesize astrocytic or oligodendrocytic-specific proteins. Moreover, differentiated RN33B cells returned to 33C re-express T antigen, but do not de-differentiate or begin dividing. Immunohistochemical and Northern blot analysis revealed high levels of low affinity NGF receptor protein and mRNA in differentiated RN33B cells. PCR analysis demonstrated the presence of trkB, but not trkA or trkC, mRNA in both undifferentiated and differentiated RN33B cells. The cells can be used as a model of neuronal differentiation in vitro.
Complete Growth Medium The base medium for this cell line is ATCC-formulated DMEM:F12 Medium Catalog No. 30-2006. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
Subculturing
Protocol:
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  3. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 33C to facilitate dispersal.
  4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
    An inoculum of 5 X 10(3) to 8 X 10(3) viable cells/sq. cm is recommended.
  6. Incubate cultures at 33C.
Interval: Maintain cultures at a cell concentration between 3 X 10(4) and 1 X 10(5) cells/sq. cm
Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:6 is recommended
Medium Renewal: Two to three times weekly
Cryopreservation
Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 33.0°C
Temperature Effects
Restrictive temperature: 37 to 39°C cells differentiate into neurons
Permissive temperature: 33°C cells proliferate
Y
Population Doubling Time 20 hours
Name of Depositor SR Whittemore
Year of Origin February, 1988
References

Whittemore SR, White LA. Target regulation of neuronal differentiation in a temperature-sensitive cell line derived from medullary raphe. Brain Res. 615: 27-40, 1993. PubMed: 8364724

Shihabuddin LS, et al. The adult CNS retains the potential to direct region-specific differentiation of a transplanted neuronal precursor cell line. J. Neurosci. 15: 6666-6678, 1995. PubMed: 7472427

Wojciechowski AB, et al. Long-term survival and glial differentiation of the brain-derived precursor cell line RN33B after subretinal transplantation to adult normal rats. Stem Cells 20: 163-173, 2002. PubMed: 11897873

Warfvinge K, et al. Retinal integration of grafts of brain-derived precursor cell lines implanted subretinally into adult, normal rats. Exp. Neurol. 169: 1-12, 2001. PubMed: 11312552

Basic Documentation
Other Documentation
References

Whittemore SR, White LA. Target regulation of neuronal differentiation in a temperature-sensitive cell line derived from medullary raphe. Brain Res. 615: 27-40, 1993. PubMed: 8364724

Shihabuddin LS, et al. The adult CNS retains the potential to direct region-specific differentiation of a transplanted neuronal precursor cell line. J. Neurosci. 15: 6666-6678, 1995. PubMed: 7472427

Wojciechowski AB, et al. Long-term survival and glial differentiation of the brain-derived precursor cell line RN33B after subretinal transplantation to adult normal rats. Stem Cells 20: 163-173, 2002. PubMed: 11897873

Warfvinge K, et al. Retinal integration of grafts of brain-derived precursor cell lines implanted subretinally into adult, normal rats. Exp. Neurol. 169: 1-12, 2001. PubMed: 11312552