Conditionally immortalized colonic epithelial cell line from a Ptk6 null mouse that polarizes and differentiates in vitro.
OBJECTIVE
PTK6 is an intracellular src-related tyrosine kinase that regulates differentiation in the intestine, where knockout animals have increased proliferative activity and growth characteristics. To explore the phenotype further we attempted to establish epithelial cell lines from the intestinal mucosa.


METHODS
We mated Ptk6 null mice with a tsSV40 large T transgenic mouse (Immortomouse) to obtain null mice carrying the SV40 gene. Intestinal tissues from these mice were cultured.


RESULTS
We established a Ptk6 null epithelial cell line from the colonic mucosa. Consistent with a role of Ptk6 in cell differentiation, these cells have the characteristics of a stable progenitor cell. In monolayer culture, the cells form domes in the monolayer when confluent. When cultured on Transwell filters, the cells polarize and form an electrically resistant barrier. Formation of tight junctions was confirmed by demonstrating expression of ZO1 and occludin at the apical junctions, whereas E-cadherin localized to the basolateral membrane. When cultured in collagen gel, the Ptk6 null cells form complex organoids, some of which resemble cups of cells. These organoids contain cells with differentiated phenotypes. Using immunohistochemistry and confocal microscopy we have been able to identify villin-positive (absorptive cells) and a small percentage of mucin-containing cells (goblet cells) and chromogranin A-positive cells (endocrine cells).


CONCLUSIONS
This conditionally immortalized cell line represents an excellent cell culture model system for exploring the mechanisms of cell function and epithelial differentiation in the colonic mucosa.
Whitehead
Robert H
RH
Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA. robert.whitehead@vanderbilt.edu
Robinson
Pamela S
PS
Williams
Janice A
JA
Bie
Wenjun
W
Tyner
Angela L
AL
Franklin
Jeffrey L
JL
D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D016764:Cell Polarity; D049109:Cell Proliferation; D048430:Cell Shape; D053379:Chromogranin A / Q000378:metabolism; D003106:Colon / Q000166:cytology / Q000201:enzymology*; D017097:Electric Impedance; D004847:Epithelial Cells / Q000201:enzymology*; D007413:Intestinal Mucosa / Q000166:cytology / Q000473:pathology*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000378:metabolism; D009077:Mucins / Q000378:metabolism; D062793:Occludin; D009940:Organoids; D010641:Phenotype; D010750:Phosphoproteins / Q000378:metabolism; D011505:Protein-Tyrosine Kinases; D019108:Tight Junctions / Q000378:metabolism; D013997:Time Factors; D062826:Zonula Occludens-1 Protein; D019061:src-Family Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
D015820:Cadherins; D053379:Chromogranin A; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009077:Mucins; D062793:Occludin; C568986:Ocln protein, mouse; D010750:Phosphoproteins; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; C026615:villin; D011505:Protein-Tyrosine Kinases; C000598269:Ptk6 protein, mouse; D019061:src-Family Kinases