Cloning differentially regulated genes from chondrocytes using agarose gel differential display.
The technique of RNA differential display has been used extensively to clone differentially expressed genes from a wide variety of cells and tissues. Recently, a simplified method of cloning differential display products, separated on agarose gels, was described. Here we report an adaption of this method, using total RNA, to clone differentially expressed genes. The approach is simple and rapid, and requires only small quantities of total RNA. Utilising this approach, we have cloned three differentially regulated genes from chondrocytes stimulated to hypertrophy in vitro, and confirmed their pattern of expression by Northern blotting. These gene fragments were sequenced and found to correspond to known genes, although only one has previously been isolated from chondrocytes.
Jefferies
D
D
Roslin Institute, Edinburgh, Midlothian, UK. david.jefferies@bbsrc.ac.uk
Botman
M
M
Farquharson
C
C
Lester
D
D
Whitehead
C C
CC
Thorp
B H
BH
Houston
B
B
D000818:Animals; D030161:Avian Proteins; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics; D002478:Cells, Cultured; D002645:Chickens; D019902:Chondrocytes / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D050556:Fatty Acid-Binding Proteins; D005786:Gene Expression Regulation; D007633:Keratins / Q000235:genetics; D054834:Lipocalins; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods
D030161:Avian Proteins; D002352:Carrier Proteins; C066299:Ch21 protein, chicken; D017931:DNA Primers; D050556:Fatty Acid-Binding Proteins; D054834:Lipocalins; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; C097184:bkj protein, Coturnix japonica; D007633:Keratins