In vitro cDNA amplification from individual intestinal crypts: a novel approach to the study of differential gene expression along the crypt-villus axis.
The mouse small intestine is lined with a monolayer of continuously renewing epithelial cells. Cells of four distinct epithelial lineages are derived clonally from the stem cell zone, located near the crypt base, from which cells differentiate and migrate to the villus tip. The kinetics of the multilineage process are well understood. However, the molecular mechanisms underlying gene expression during lineage commitment and cell proliferation and differentiation remain obscure. A novel approach to the problem is presented here. Single intact epithelial crypts were isolated by incubation in ethylenediaminetetraacetic acid and mechanical vibration of everted mouse intestinal or colonic segments. Crypts isolated in this manner were suitable for mRNA-directed polymerase chain reaction, thus generating crypt epithelium-specific cDNA. The fidelity of transcript amplification was confirmed by Southern blot hybridization with cloned intestinal transcripts. To demonstrate the potential utility of crypt-specific cDNA, the amplified transcripts from a single jejunal crypt were used to construct a cDNA library, characterization of which revealed a high representation of cryptdin-1-related transcripts. This study presents a technique which will facilitate comprehensive analyses of gene expression in the differentiating mammalian intestine.
Cano-Gauci
D F
DF
Ontario Cancer Institute, University of Toronto, Canada.
Lualdi
J C
JC
Ouellette
A J
AJ
Brady
G
G
Iscove
N N
NN
Buick
R N
RN
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D002469:Cell Separation / Q000379:methods; D003106:Colon / Q000502:physiology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA / Q000235:genetics; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D015870:Gene Expression; D015723:Gene Library; D007413:Intestinal Mucosa / Q000502:physiology*; D007421:Intestine, Small / Q000033:anatomy & histology / Q000502:physiology*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011498:Protein Precursors / Q000235:genetics; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
D002352:Carrier Proteins; C082667:Fabp5 protein, mouse; C086936:Fabp7 protein, mouse; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D011506:Proteins; D012333:RNA, Messenger; C059569:cryptdin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA