Immortalisation, characterisation and differentiation of temperature sensitive cell lines from the Olfactory Neuroepithelium

Embryonic olfactory neuroepithelium provides a useful experimental system for the study of olfactory neurogenesis. As a substrate for experimental neural cell biology, olfactory neuroepithelium is of unique interest since, unlike other neural cells, olfactory neurons are continually replaced - a feature that is dictated by their direct exposure to the damaging external environment. Basal cells in the olfactory placode are the source of this replacement. Each olfactory neuron expresses only one or a few of the many olfactory receptors that are encoded by the large array of olfactory genes. Despite this limited cellular display of receptors, vertebrates are able to distinguish many thousands of different odorants, implying a complicated need for perceptive neurological processing of signals coming from individual olfactory neurons. To study the events that take place during the differentiation of neuronal precursors - a process that sustains a diverse receptor repertoire - I felt that lines of conditionally immortalised cells that could be induced to differentiate would provide useful reagents. In this thesis I describe my successful attempts to immortalise olfactory cell lines from the neuroepithelium of E 10.5 mouse embryos. I used a conditionally immortalising retrovirus that included the coding sequence for the temperature-sensitive SY 40 large T antigen. Integration of this retrovirus into the genome of cells allowed continuous proliferation at the permissive temperature of 33°C. A shift to the nonpermissive temperature of 39°C inactivated the SV40 large T antigen, the cells ceased proliferation and differentiation commenced. Sixty cell lines were derived of which four were chosen for further characterisation. These four cell lines (OP6, OP27, OP47 and OP55) were clonally derived and were immortalised rather than transformed. They continued to express the SV40 large T antigen at 33°C but lost expression at 39°C concomitant with cessation of proliferation. When the OP cells were shifted to 39°C in the absence or presence of the morphogen, retinoic acid, morphological changes ensued that were consistent with the development of neuronal characteristics. The OP6, OP27 and the OP47 cells became phase-bright with neuritic extensions. The OP55 cells were the exception in that they did not develop extensions but instead differentiated to form compact epithelial islands when grown in DM-10 medium but not in RA medium. Differentiation of the OP cells at 39°C was further documented by the induced expression of a number of markers demonstrated by RT-PCR and/or immunocytochemistry. The OP cells differentiated at 39°C in DM-10 and in retinoic acid-containing medium to express olfactory receptor transcripts. Cloning and sequencing showed that each cell line expressed a single receptor type but that different receptors were expressed by different cell lines. Sequencing revealed that the receptors cloned from the OP27 cells were 98% homologous to the mouse-M65 olfactory receptor whereas OP55 had greatest homology to rat-Olf3 olfactory receptor. The transcripts induced in OP6 and the OP47 cells showed greatest homology with Gus58 - a taste receptor homologous to olfactory receptors. Sequences obtained from OP6, OP47 and OP55 cells were not 100% identical to published receptors and could thus represent members of different subfamilies. Interestingly, induced OP55 cells also expressed mRNA for clusterin - a molecule that has no homology with olfactory receptor transcripts but is involved in differentiation during embryogenesis.

Grantor dc:publisher.institution

Department of Molecular and Cell Biology

Year dc:date.issued

1999

Author dc:creator

• Boolay, Sihaam

Advisor dc:contributor.advisor

• Illing, Nicola

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/11427/22552

OAI identifier oai:identifier

oai:open.uct.ac.za:11427/22552