Establishment of renal proximal tubule cell lines by targeted oncogenesis in transgenic mice using the L-pyruvate kinase-SV40 (T) antigen hybrid gene

1993 ◽  
Vol 104 (3) ◽  
pp. 695-704 ◽  
Author(s):  
N. Cartier ◽  
R. Lacave ◽  
V. Vallet ◽  
J. Hagege ◽  
R. Hellio ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Proximal Tubule ◽  
Pyruvate Kinase ◽  
Cell Lines ◽  
Cultured Cells ◽  
Simian Virus ◽  
T Antigen ◽  
Regulatory Sequence ◽  
Proximal Tubule Cell ◽  
Proximal Tubule Cells

Targeted oncogenesis allowed us to obtain two cell lines which have been derived from the proximal tubule of kidney from transgenic mice harbouring the simian virus (SV40) large T and small t antigens placed under the control of the 5′ regulatory sequence from the rat L-type pyruvate kinase (L-PK) gene. The cell lines (PKSV-PCT and PKSV-PR cells) were derived from early (PCT) and late (Pars Recta, PR) microdissected proximal tubules grown in D-glucose-enriched medium. In such conditions of culture, both cell lines exhibited L-PK transcripts, a stable expression of SV40-encoded nuclear large T antigen, a prolonged life span but failed to induce tumors when injected sub-cutaneously into athymic (nu-nu) mice. Confluent cells, grown on plastic support or porous filters, were organized as monolayers of polarized cuboid cells with well developed apical microvilli and formed domes. Both cell lines exhibited morphological features of proximal tubule cells with villin located in the apical brush-border and substantial amounts of hydrolase activity. By immunofluorescence studies using specific antibodies, aminopeptidase N appeared restricted to the apical microvillar domain, whereas the H2 histocompatibility antigen was distributed in the cytoplasm and lateral membranes. These results demonstrate that the proximal morphological phenotype has been fully preserved in these cultured cells derived from tissue-specific targeted oncogenesis in transgenic mice.

Functional properties of proximal tubule cell lines derived from transgenic mice harboring L-pyruvate kinase-SV40 (T) antigen hybrid gene

1993 ◽  
Vol 104 (3) ◽  
pp. 705-712 ◽  
Author(s):  
R. Lacave ◽  
M. Bens ◽  
N. Cartier ◽  
V. Vallet ◽  
S. Robine ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Pyruvate Kinase ◽  
Cell Lines ◽  
Cultured Cells ◽  
Functional Characterization ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Regulatory Sequence ◽  
Proximal Tubule Cell ◽  
Sodium Dependent

This study describes the functional characterization of two cell lines derived from the proximal convoluted (PKSV-PCT cells) and proximal straight (PKSV-PR) tubules microdissected out from kidneys of transgenic mice harboring the simian virus 40 (SV40) large T and small t antigens placed under the control of the rat L-type pyruvate kinase (L-PK) 5′ regulatory sequence. Both cell lines exhibited cellular cyclic AMP stimulated by parathormone (PTH) and calcitonin (CT) and a sodium-dependent glucose transporter. Uptake of the fluid-phase marker [3H]inulin showed that both cell lines grown on filters exhibited biphasic apical and basolateral endocytic rates. Results from Northern blot analysis indicate that the expression of the T antigen gene (Tag) is dependent on the concentration of D-glucose in the medium and show that the L-PK construct has maintained its capacity for up- or down-regulation by carbohydrates. Replacement of D-glucose by neoglucogenic substrates (lactate, oxaloacetate) blunted the expression of Tag transcripts and induced arrest of cell growth. Compared to cell grown in D-glucose-enriched medium, the hormonal sensitivities to PTH and CT and the sodium-dependent glucose uptake were unchanged whereas quiescent cells exhibited increased hydrolase content. Thus the proximal function has been preserved in these cultured cells derived from tissue-specific targeted oncogenesis in transgenic mice. As the expression of Tag transcripts is controlled by D-glucose, the structural and physiological characteristics of these cell lines can be studied in either quiescent or active growth conditions.

Na+/H+ exchanger activity and phosphorylation in temperature-sensitive immortalized proximal tubule cell lines derived from the spontaneously hypertensive rat

2000 ◽  
Vol 98 (4) ◽  
pp. 409-418 ◽  
Author(s):  
Leong L. NG ◽  
Sonja JENNINGS ◽  
Joan E. DAVIES ◽  
Paulene A. QUINN
Keyword(s):  
Proximal Tubule ◽  
Cell Lines ◽  
Western Blotting ◽  
Spontaneously Hypertensive Rat ◽  
Proximal Tubule Cell ◽  
Permissive Temperature ◽  
Proximal Tubule Cells ◽  
Spontaneously Hypertensive ◽  
Hypertensive Rat ◽  
Tubule Cells

Freshly isolated proximal tubules from the spontaneously hypertensive rat (SHR) demonstrate elevated Na+/H+ exchanger (NHE) activity, but the underlying mechanism is unclear. Because of the difficulties in preparing sufficient numbers of proximal tubule cells for detailed biochemical studies, we have generated cell lines from SHR and Wistar–Kyoto rat (WKY) proximal tubule cells. Cell lines were obtained by transforming the cells with an origin-defective mutant of simian virus 40 encoding a heat-labile T antigen (tsA58 transformant). Such cells proliferate at the permissive temperature of 33 °C, but growth is abolished at the restrictive temperature of 39 °C. The predominant NHE isoform expressed was isoform 1, as determined by sensitivity to HOE-694 (3-methylsulphonyl-4-piperidinobenzoyl guanidine) and Western blotting using specific polyclonal antisera to NHE-1. NHE-3 protein was also present. Northern blots of poly(A) mRNA extracts of the cell lines revealed a low abundance of transcripts for NHE-2, -3 and -4, with no systematic difference between the lines. Although the intracellular pH was similar in the SHR and WKY lines, HOE-694-sensitive H+ efflux due to NHE-1 was substantially elevated in SHR lines compared with WKY lines (95.0±2.8 and 39.9±5.7 mmol·min-1·l-1 respectively; P < 0.001; n = 6). H+ efflux due to non-Na+-dependent mechanisms were similar in lines from the two strains. Western blotting revealed that NHE-1 density was also very similar in SHR and WKY lines, and subcellular fractionation of homogenates indicated that NHE-1 was localized predominantly to plasma membranes. Thus the turnover number of NHE-1 was increased. Immunoprecipitation of 32P-labelled phosphoproteins from these lines demonstrated an approximately 2-fold higher degree of phosphorylation of NHE-1 in SHR compared with WKY lines. These cell lines form a useful model for defining the biochemical mechanisms leading to the NHE-1 phenotype in the SHR kidney, in addition to investigations of other SHR phenotypic markers.

Transimmortalized mouse intestinal cells (m-ICc12) that maintain a crypt phenotype

1996 ◽  
Vol 270 (6) ◽  
pp. C1666-C1674 ◽  
Author(s):  
M. Bens ◽  
A. Bogdanova ◽  
F. Cluzeaud ◽  
L. Miquerol ◽  
S. Kerneis ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Regulatory Sequence ◽  
Transmembrane Conductance Regulator ◽  
Large T Antigen ◽  
Transmembrane Conductance ◽  
Immunoglobulin Receptor ◽  
Immortalized Cells

This study describes the properties of a clone of immortalized cells (m-ICc12 cells) derived from the bases of small intestinal villi from 20-day-old fetuses of L-type pyruvate kinase (L-PK)/ TAg1 transgenic mice. The mice harbor the simian virus 40 large T antigen under the control of the 5' regulatory sequence from the L-PK gene. m-ICc12 cells expressed nuclear large T antigen, had a prolonged life span, and were nontumorigenic when injected into nude mice. They formed confluent monolayers of cuboid cells separated by tight junctions, developed dense, short apical microvilli, and formed domes. They also possessed cytokeratins, villin, aminopeptidase N, dipeptidyl-peptidase IV, and glucoamylase and retained crypt cell features, including intracellular sucrase isomaltase and alpha-L-fucose glycoconjugates accumulation and expression of the polymeric immunoglobulin receptor and the cystic fibrosis transmembrane conductance regulator gene. Thus the m-ICc12 cell line obtained by targeted oncogenesis in transgenic mice maintained in culture several important properties and differentiated functions of intestinal crypt cells.

Sign in / Sign up

Export Citation Format

Share Document