A method for analysis of gene expression in isolated mouse photoreceptor and Müller cells.
OBJECTIVE
Molecular analysis of complex phenomena, such as selective death of photoreceptors and their rescue by neuro-protective agents, has been hindered by limitations of techniques for investigating gene expression in individual cells within a heterogeneous tissue such as the retina. The purpose of this study was to develop methods to assess gene expression in single retinal cells.


METHODS
Individual cells from papain-dissociated mouse retinae were captured with micropipettes and identified by morphology and by immunocytochemistry. Single cell cDNA libraries were generated by poly-d(T)-primed reverse transcription, poly-d(A) tailing of first strand cDNA, and en masse PCR-amplification using a custom made oligo-d(T). PCR was used to investigate gene expression in cDNAs from individual cells.


RESULTS
Dissociated rod and Müller glia cells maintained their morphology, which correlated with their immunocytochemical properties. RPE cells were recognized by their pigmentation. With the exception of bipolar cells, non-photoreceptor neurons were only identifiable by immunocytochemistry. Abundant cDNA could be synthesized from each individual cell. Cell-specific "markers" were detected by PCR almost exclusively in the predicted cell types. The expression of neurotrophic factor receptors was consistent with previous biological studies.


CONCLUSIONS
These studies establish a method to compare, investigate, and analyze gene expression in individual cells of the retina.
Wahlin
Karl J
KJ
Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287-9257, USA.
Lim
Lynette
L
Grice
Elizabeth A
EA
Campochiaro
Peter A
PA
Zack
Donald J
DJ
Adler
Ruben
R
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D018076:DNA, Complementary / Q000235:genetics; D005136:Eye Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology; D015723:Gene Library; D051379:Mice; D008810:Mice, Inbred C57BL; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology / Q000378:metabolism*
D015415:Biomarkers; D018076:DNA, Complementary; D005136:Eye Proteins