Intestinal lactase as an autologous beta-galactosidase reporter gene for in vivo gene expression studies.
Intestinal lactase has potential as an autologous beta-galactosidase reporter gene for long-term gene expression studies in vivo, using chromogenic, luminescent, and fluorogenic substrates developed for Escherichia coli beta-galactosidase. In normal rat tissues, reactivity with a chromogenic fucopyranoside (X-Fuc, the preferred substrate of lactase) was present only at the lumenal surface of small intestine epithelial cells. Full-length lactase (domains I-IV), mature lactase (domains III and IV), and a cytosolic form of mature lactase (domains III and IV, without the signal sequence or transmembrane region) were evaluated. Transfection of HuH-7 cells in vitro, and hydrodynamic gene delivery to the liver in vivo, resulted in excellent gene expression. The full-length and mature (homodimeric, membrane-bound) forms reacted strongly with X-Fuc but not with the corresponding galactopyranoside (X-Gal). However, the presumptively monomeric cytosolic lactase unexpectedly reacted equally well with both substrates. The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was cleaved by cytosolic lactase, but not by full-length or mature lactase. Full-length lactase, when expressed ectopically in hepatocytes in vivo, localized exclusively to the bile canalicular membrane. Intestinal lactase is highly homologous in mice, rats, and humans and has considerable potential for evaluating long-term gene expression in experimental animals and the clinic.
Salehi
Siamak
S
Department of Hepatology and Transplantation, King's College London School of Medicine, James Black Centre, London SE5 9NU, United Kingdom.
Eckley
Lorna
L
Sawyer
Greta J
GJ
Zhang
Xiaohong
X
Dong
Xuebin
X
Freund
Jean-Noel
JN
Fabre
John W
JW
D000818:Animals; D045744:Cell Line, Tumor; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D043322:Lactase / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D051381:Rats; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D043322:Lactase; D001616:beta-Galactosidase