NIT-2 (ATCC® CRL-2364)

Organism: Mus musculus, transgenic for SV40 large T antigen, mouse, transgenic for SV40 large T antigen  /  Cell Type: beta cell  /  Disease: adenoma; carboxypeptidase E defective

Organism Mus musculus, transgenic for SV40 large T antigen, mouse, transgenic for SV40 large T antigen
Cell Type beta cell
Product Format frozen
Morphology epithelial
Culture Properties adherent
Biosafety Level 2 Cells contain polyomavirus DNA sequences
Disease adenoma; carboxypeptidase E defective
Age 10 week old
Gender male
Applications
The NIT-2 cell line was derived from the pancreatic beta cells of Cpe(fat)/Cpe(fat) mice by crossing C57BLKS/J-Cpe(fat)/+ mice with NOD/Lt-Tg(RIPTag)1Lt mice.
NOD/Lt-Tg(RIPTag)1Lt mice are transgenic for the SV40 large T antigen under the control of a rat insulin promoter, and spontaneously develop beta adenomas.
Pro-CPE, but not the mature form of CPE, is present in NIT-2 cells, and neither pro-CPE nor CPE are secreted into the medium.
The secretion of insulin/proinsulin from NIT-2 cells is significantly elevated by secretagogues, indicating that CPE is not required for sorting proinsulin into the regulated pathway.
Derivation
The NIT-2 cell line was derived from the pancreatic beta cells of Cpe(fat)/Cpe(fat) mice by crossing C57BLKS/J-Cpe(fat)/+ mice with NOD/Lt-Tg(RIPTag)1Lt mice.
The NIT-2 cell line was cultured from adenomatous islets obtained from a 10 week old F2 male and was compared with the NIT-1 cell line (ATCC-CRL-2055) previously developed from mice with wild-type CPE.
Clinical Data
The NIT-2 cell line was cultured from adenomatous islets obtained from a 10 week old F2 male and was compared with the NIT-1 cell line (ATCC-CRL-2055) previously developed from mice with wild-type CPE.
male
Comments
The NIT-2 cell line was derived from the pancreatic beta cells of Cpe(fat)/Cpe(fat) mice by crossing C57BLKS/J-Cpe(fat)/+ mice with NOD/Lt-Tg(RIPTag)1Lt mice.
NOD/Lt-Tg(RIPTag)1Lt mice are transgenic for the SV40 large T antigen under the control of a rat insulin promoter, and spontaneously develop beta adenomas.
Carboxypeptidase E is required for complete conversion of proinsulin to mature insulin. A spontaneous point mutation in the coding region of the carboxypeptidase E (CPE) gene in Cpe(fat)/Cpe(fat) mice affects proinsulin processing.
The NIT-2 cell line was cultured from adenomatous islets obtained from a 10 week old F2 male and was compared with the NIT-1 cell line (ATCC-CRL-2055) previously developed from mice with wild-type CPE.
Electron microscopy of the cultured NIT-2 showed increased numbers of enlarged and electron-lucent granules compared with NIT-1 cells.
Pro-CPE, but not the mature form of CPE, is present in NIT-2 cells, and neither pro-CPE nor CPE are secreted into the medium.
Proinsulin is less extensively processed in NIT-2 than in NIT-1 cells, indicating that the Cpe(fat)mutation affects both the endopeptidase and carboxypeptidase reactions.
The secretion of insulin/proinsulin from NIT-2 cells is significantly elevated by secretagogues, indicating that CPE is not required for sorting proinsulin into the regulated pathway.
Complete Growth Medium Ham's F12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 90%; heat-inactivated dialyzed fetal bovine serum, 10%.
Subculturing
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:3 is recommended
Medium Renewal: Twice per week
Subcultures are prepared using a cell dissociation buffer (an enzyme free Hanks' based solution; Catalog number: 13150-016 available from GIBCO).
Remove the medium from the culture flask, add 2 ml of cell dissociation buffer per 25 sq. cm flask (5 ml per 75 sq. cm. flask and gently rock the flask at room temperature for 1 to 2 minutes to bathe the cells in the buffer.
Aspirate the solution and discard. Allow the flask to sit at room temperature for 3 to 4 additional minutes (total time from initial addition of cell dissociation buffer is approximately 5 minutes).
Firmly tap the flask against the palm of the hand to dislodge cells.
Add 5 ml of fresh medium per 25 sq. cm. flask (10 ml per 75 sq. cm. flask) and triturate up and down directing the stream along the bottom of the flask to dislodge the cells and break up some of the clumps.
Add fresh medium, aspirate and dispense into new flasks.
Cryopreservation
Culture medium, 95%; DMSO, 5%
Culture Conditions
Temperature: 37.0°C
Name of Depositor EH Leiter
References

Varlamov O, et al. Beta-cell lines derived from transgenic Cpe(fat)/Cpe(fat) mice are defective in carboxypeptidase E and proinsulin processing. Endocrinology 138: 4883-4892, 1997. PubMed: 9348219

Naggert JK, et al. Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity. Nat. Genet. 10: 135-142, 1995. PubMed: 7663508

Basic Documentation
References

Varlamov O, et al. Beta-cell lines derived from transgenic Cpe(fat)/Cpe(fat) mice are defective in carboxypeptidase E and proinsulin processing. Endocrinology 138: 4883-4892, 1997. PubMed: 9348219

Naggert JK, et al. Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity. Nat. Genet. 10: 135-142, 1995. PubMed: 7663508