A subtractive PCR-based cDNA library made from fetal thymic stromal cells.
We describe our initial approach to clone and characterize genes expressed preferentially in thymic stromal cells, in an attempt to generate molecular reagents to study the role of these cells in thymopoiesis and thymic function. Thymic stromal cells were prepared from fetal thymic organ cultures by treating them with 2-deoxyguanosine and depleting the remaining hematopoietic cells with anti-CD45 antibody. A cDNA library was then prepared after subtraction and amplification by PCR. The cloned inserts were sequenced and compared for homology with known genes in the data base. Unidentified cDNAs were then examined for expression in normal and SCID thymus and in a set of SV40-transformed thymic epithelial cell lines, by Northern blotting and a dot blot assay. In this report we describe the development of the library and present a general description of the genes identified from the initial 249 cDNAs sequenced. Among these, a relatively high percentage (55%) do not show any homology to previously identified genes. Several genes with a limited expression pattern were selected for further study.
Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0420, USA.
M G
MG
Kim
C
C
Chen
F A
FA
Flomerfelt
R N
RN
Germain
R H
RH
Schwartz
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification*; D003849:Deoxyguanosine / Q000494:pharmacology; D005260:Female; D005333:Fetus; D015870:Gene Expression; D005796:Genes; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009924:Organ Culture Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism; D017154:Stromal Cells / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013950:Thymus Gland / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D003849:Deoxyguanosine