HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ontsouka, E. C. Articles by Blum, J. W. Search for Related Content PubMed PubMed Citation Articles by Ontsouka, E. C. Articles by Blum, J. W.

Real-Time PCR Quantification of Bovine Lactase mRNA: Localization in the Gastrointestinal Tract of Milk-Fed Calves^*

E. C. Ontsouka¹, B. Korczak², H. M. Hammon^1, and J. W. Blum¹

¹ Division of Animal Nutrition and Physiology, Institute of Animal Genetics, Nutrition, and Housing, and
² Institute of Veterinary Bacteriology, Vetsuisse Faculty University of Berne, CH-3012 Berne, Switzerland

Corresponding author: J. W. Blum; e-mail: juerg.blum{at}itz.unibe.ch.

Lactase is a disaccharidase that is present in the brush-border membrane of the small intestine, hydrolyzes lactose to glucose and galactose, and is therefore important in milk-fed animals. Assays based on quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) in the bovine species have not yet been described. Therefore, we have developed an RT-PCR assay for the quantification of lactase mRNA levels and have tested its suitability in the bovine gastrointestinal tract of seven 5-d-old milk-fed calves. Primers for RT-PCR amplification of bovine lactase mRNA were designed in the 100% identical regions of species (rats, rabbits, humans) from which lactase sequences were available. Lactase mRNA was expressed relative to mean levels of 4 housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase, ß-actin, ubiquitin, and 18S). The presence of lactase mRNA along the entire gastrointestinal tract was evaluated in samples that consisted of whole gut walls (mucosa plus submucosa). Furthermore, mRNA levels of lactase were measured in fractionized layers of jejunal and ileal mucosa (mainly containing villus tips or crypts) and ileal lamina propria (mainly containing Peyer’s patches). Agarose gel electrophoresis of the lactase PCR product revealed a single band that corresponded to the single-amplified product as predicted by the melting curve analysis of the PCR. The amplified partial-bovine lactase sequence showed 87% similarity with human and rabbit sequences and 82% similarity with the rat sequence. Lactase mRNA was present in whole walls (consisting of mucosa and submucosa) of the entire small intestine, but was absent in esophagus, rumen, fundus, pylorus, and colon. Furthermore, lactase mRNA was detected in fractionized villus and crypt layers of jejunum and ileum, but levels were higher in the jejunum in villus than in crypt fractions. No lactase mRNA was detectable in the lamina propria fraction of the ileum containing mainly Peyer’s patches. In conclusion, the developed RT-PCR method allows study of lactase mRNA levels.

Key Words: gastrointestinal tract • mRNA • lactase • calf

Abbreviation key: BrdU = 5'-bromo-2-deoxyuridine, CP = crossing point, GAPDH = glyceraldehyde-3-phosphate dehydrogenase, HKG = housekeeping gene, LP = lamina propria, PP = Peyer’s patches, RT-PCR = reverse transcription-polymerase chain reaction, SI = small intestine.