Isolation, culture and adenoviral transduction of parietal cells from mouse gastric mucosa.
Here we describe a method for the isolation of intact gastric glands from mice and primary culture and transfection of mouse gastric epithelial cells. Collagenase digestion of PBS-perfused mouse stomachs released large intact gastric glands that were plated on a basement membrane matrix. The heterogeneous gland cell cultures typically contain approximately 60% parietal cells. Isolated mouse parietal cells remain viable in culture for up to 5 days and react strongly with an antibody specific to the gastric H(+)/K(+) ATPase. Isolated intact mouse gastric glands and primary cultures of mouse parietal cells respond to the secretagogue, histamine. Typical morphological changes from a resting to an acid-secreting active parietal cell were observed. In resting cultures of mouse parietal cells, the H(+)/K(+) ATPase displayed a cytoplasmic punctate staining pattern consistent with tubulovesicle element structures. Following histamine stimulation, an expansion of internal apical vacuole structures was observed together with a pronounced redistribution of the H(+)/K(+) ATPase from the cytoplasm to the apical vacuoles. A reproducible procedure to express genes of interest exogenously in these cultures of mouse parietal cells was also established. This method combines recombinant adenoviral transduction with magnetic field-assisted transfection resulting in approximately 30% transduced parietal cells. Adenoviral-transduced parietal cells maintain their ability to undergo agonist-induced activation. This protocol will be useful for the isolation, culture and expression of genes in parietal cells from genetically modified mice and as such will be an invaluable tool for studying the complex exocytic and endocytic trafficking events of the H(+)/K(+) ATPase which underpin the regulation of acid secretion.
Gliddon
Briony L
BL
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Victoria, Australia.
Nguyen
Nhung V
NV
Gunn
Priscilla A
PA
Gleeson
Paul A
PA
van Driel
Ian R
IR
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D005753:Gastric Mucosa / Q000166:cytology* / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D010295:Parietal Cells, Gastric / Q000166:cytology* / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods*
D011994:Recombinant Proteins