اشتقاق خطوط التوتة تي خلية سرطان الغدد الليمفاوية من أجهزة الصراف الآلي -- / --</sup و البروتين p53 -- / --</sup الفئران
Summary
In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.
Abstract
Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.
Protocol
Discussion
In this protocol, we provide detailed procedures to establish murine T-cell lines from two different mouse genotypes; Atm-/- [FVB/N-Atmtm1Led/J] and p53-/- [129/S6-Trp53tm1Tyj/J]. By using this protocol, a total of 6 (out of 7 attempts) Atm-/- and three (out of 5 attempts) p53-/- murine T-cell lines have been established in our laboratory. Representative data demonstrating that cell line DWJ-Atm-1, an<e…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors wish to thank Dr. Boris Reizis from the Department of Microbiology and Immunology at Columbia University for helpful discussions. We also wish to thank Dr. Margaret Bynoe, Dr. Rodman Getchell and Deequa Mahamed from the Department of Microbiology and Immunology at the College of Veterinary Medicine, Cornell University for technical assistance with flow cytometry and Lu Huang for assistance with video editing. The authors also wish to thank the members of Duhamel and Weiss laboratories for their critical review of the manuscript and video production, and Stephanie Yazinski from the Weiss laboratory for breeding and genotyping p53-/- mice. Experiments on animals were performed in accordance with the guidelines and regulations set forth by Cornell University Institutional Animal Care and Use Committee. This research was supported in part by funds provided by the US Department of Agriculture, Cooperative State Research, Education, and Extension Service, Animal Health and Disease Research Program (to G.E.D. and R.S.W.) and NIH grants R03 HD058220 and R01CA108773 (to R.S.W). The video was produced using in-house facilities by the personnel from the Duhamel & Weiss laboratories.
Materials
- Ice bucket
- 70% ethanol
- Dissection pad consisting of foam block covered with aluminum foil and pins
- Two sets of small sterile scissors and forceps
- Sterile 150 mm2 Petri dish (Thermo Fisher Scientific, Waltham, MA; cat. #08-757-13)
- Sterile 18 gauge needles
- 50 mL conical tubes (Corning Inc., Corning NY; cat # 430829)
- 10% buffered formalin
- Tissue culture flasks
- T-25 (TPP, Trasadingen, Switzerland; cat # 90025)
- T-75 (TPP, Trasadingen, Switzerland; cat # 90075)
- 6-well plates (Corning Inc., Corning NY; cat # 3506)
- Sterile phosphate buffered saline pH 7.2 (PBS)
- Culture medium
- RPMI 1640 with 25 mM HEPES, 200 mM L-glutamine (Lonza, Walkersville, MD; cat#12-115F) supplemented with the following:
- 10% heat inactivated (56°C, 30 min) fetal bovine serum (FBS; Hyclone, Logan, UT; cat# SH30088.03)
- 1% penicillin and streptomycin (Mediatech, Manassas,VA; cat# 30-002-CI)
- 1% nonessential amino acids (Mediatech; cat#25-025-CI)
- 55 mM 2β-mercaptoethanol (Sigma, St Louis, MO; cat#M752)
- RPMI 1640 with 25 mM HEPES, 200 mM L-glutamine (Lonza, Walkersville, MD; cat#12-115F) supplemented with the following:
- Dimethyl sulfoxide (DMSO; Calbiochem, La Jolla, CA; cat # 317275)
- Antibodies (eBioScience, San Diego, CA)
- anti-CD3-FITC (cat. #11-0031-85)
- anti-CD4-APC (cat. #17-0041-83)
- anti-CD8-PE (cat.#12-081-85)
- Bovine serum albumin (BSA; Life Technologies, Grand Island, NY; cat #. 11018-017)
- Concanavalin A (Sigma cat# C5275)
- 1.8 mL freezing vials (Nunc, Roskilde, Denmark; cat# 368632)
Riferimenti
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