In situ transcription and detection of CD1a mRNA in epidermal cells: an alternative to standard in situ hybridization techniques.
To develop methods for the investigation of mRNA transcription in rare epidermal cells, we used in situ transcription to study CD1a mRNA in isolated CD1a positive cells. We chose to study this Langerhans cell marker because it is not known which epidermal cells actually produce the CD1a protein and because there is evidence that CD1a mRNA is alternately spliced, a situation which could lead to truncated or alternate protein products in CD1a surface protein negative cells. Disaggregated epidermal cells were resolved into CD1a surface protein positive and negative groups by fluorescence activated cell sorting and cytocentrifuged onto glass slides. A synthetic 52 base, CD1a specific anti-sense oligomer was hybridized to CD1a gene transcripts in these cells, and radiolabeled cDNA synthesized in situ on the oligomerprimed CD1a transcripts. The labeled cDNA fragments were visualized in the cells of origin by autoradiography, and grains per cell were counted. Sixty-eight percent of cells expressing CD1a protein contained CD1a mRNA, as evidenced by grain counts more than two standard deviations above the mean value for similar cells carried through the same procedure with a control oligomer, or the mean value of CD1a surface protein negative cells treated with the CD1a specific oligomer. Thus, it seems likely that the CD1a protein positive epidermal cells use CD1a mRNA to make their own CD1a protein, and that a truncated or masked CD1a protein is not made by CD1a negative neonatal foreskin epidermal cells. In our hands, in situ transcription is simpler and faster than standard methods of in situ hybridization with prelabeled cDNA or RNA probes. Furthermore, it can be applied to the detection of any message of known sequence. The combination of cell sorting and in situ transcription can be used to localize and quantify the expression of specific mRNA by individual cells, allowing the study of rare and difficult-to-obtain cells.
Longley
J
J
Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06511.
Merchant
M A
MA
Kacinski
B M
BM
D018949:Antigens, CD1; D000943:Antigens, Differentiation / Q000032:analysis*; D001345:Autoradiography; D000078404:Epidermal Cells; D004817:Epidermis / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D013997:Time Factors; D014158:Transcription, Genetic
D018949:Antigens, CD1; D000943:Antigens, Differentiation; D012333:RNA, Messenger