| 產品名稱 |
NIT-2 |
| 商品貨號 |
B165397 |
| Organism |
Mus musculus, transgenic for SV40 large T antigen, mouse, transgenic for SV40 large T antigen |
| Tissue |
pancreas |
| Cell Type |
beta cell |
| Product Format |
frozen |
| Morphology |
epithelial |
| Culture Properties |
adherent |
| Biosafety Level |
2 Cells contain polyomavirus DNA sequences
Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country. |
| 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. |
| Storage Conditions |
liquid nitorgen vapor phase |
| 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 |
Volumes used in this protocol are for 25 cm2 or 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
NOTE: NIT-2 cells will not form a confluent monolayer, however, they will form nice colonies of monolayered cells in a fairly dense array. When the NIT-2 colonies begin to "ball-up" slightly and show many round cells on top of the monolayers as well as floating in the media, it is time to passage them.
- Remove and discard culture medium.
- Subcultures are prepared using a cell dissociation buffer (an enzyme?free Hanks' based solution; GIBCO, Catalog #13150-016). Add 2 mL cell dissociation buffer per 25 cm. sq. flask (5 mL per 75 cm. sq. flask) and gently rock flask to bathe the cells at room temperature for 1 to 2 minutes.
- Aspirate the solution and discard.
- Allow the flask to remain at room temperature for 5 additional minutes (total time from initial addition of cell dissociation buffer approximately 7 minutes).
- FirmLy tap the flask against palm of hand to dislodge cells.
- Add 5 mL of fresh medium per 25 cm2 flask (10 mL per 75 cm2 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 appropriate aliquots of cell suspension to new culture vessels.
- Place culture vessels in incubators at 37°C.
Subcultivation Ratio: 1:2 to 1:3
Medium Renewal: Twice weekly
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.
|
| Cryopreservation |
Complete growth medium described above supplemented with 5% (v/v) DMSO. Cell culture tested DMSO is available as ATCC Catalog No. 4-X. |
| Culture Conditions |
Temperature: 37°C
Atmosphere: Air, 95%; Carbon dioxide (CO2), 5% |
| Name of Depositor |
EH Leiter |
| Deposited As |
mouse, transgenic for SV40 large T antigen |
| 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
Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.
Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.
Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.
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