Cataloging altered gene expression in young and senescent cells using enhanced differential display.
Recently, a novel PCR-based technique, differential display (DD), has facilitated the study of differentially expressed genes at the mRNA level. We report here an improved version of DD, which we call Enhanced Differential Display (EDD). We have modified the technique to enhance reproducibility and to facilitate sequencing and cloning. Using EDD, we have generated and verified a catalog of genes that are differentially expressed between young and senescent human diploid fibroblasts (HDF). From 168 genetags that were identified initially, 84 could be sequenced directly from PCR amplified bands. These sequences represent 27 known genes and 37 novel genes. By Northern blot analysis we have confirmed the differential expression of a total of 23 genes (12 known, 11 novel), while 19 (seven known, 12 novel) did not show differential expression. Several of the known genes were previously observed by others to be differentially expressed between young and senescent fibroblasts, thereby validating the technique.
Linskens
M H
MH
Geron Corporation, Menlo Park, CA 94025, USA.
Feng
J
J
Andrews
W H
WH
Enlow
B E
BE
Saati
S M
SM
Tonkin
L A
LA
Funk
W D
WD
Villeponteau
B
B
D001483:Base Sequence; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D015342:DNA Probes; D012333:RNA, Messenger