Detection of transgene product in mouse peripheral blood lymphocytes: a minimally invasive microassay.
Confirmation of transgene-coded protein expression on the surface of mouse white blood cells has traditionally involved use of spleen and lymph nodes as a cell source. However, this eliminates the mice from further analysis and increases the number of animals needed for testing. A minimally invasive method of cell collection would allow these mice to remain in the colony, thereby conserving valuable resources. The goal of the study reported here was to detect transgene expression on mouse white blood cells collected from peripheral blood. Mice over-expressing the T-cell receptor (TCR) transgene product Vbeta8.2 on the surface of T lymphocytes were compared with wild-type control mice. Mononuclear cells were counted, using a hemacytometer. Peripheral blood cells were stained with anti-Vbeta8.2 fluorescent conjugate and were evaluated, using fluorescence-activated (FA) microscopy as well as flow cytometry (fluorescence-activated cell sorting [FACS]). Mean +/- SD number of mononuclear cells was 5.34 +/- 1.72 x 10(5) cells/100 ml from TCR Tg mice and 4.99 +/- 1.99 x 10(5) cells/100 microl from control mice. Expression of transgene Vbeta8.2 was confirmed by use of FA microscopy and FACS. Mean population of lymphocytes expressing Vbeta8.2 was 43.0 +/- 12.9% from TCR Tg blood samples, and 9.4 +/- 1.0% from control blood samples. Spleen and lymph node specimens also were compared. Results of the unpaired t test and the Mann-Whitney test indicated significant difference in the amount of expressed Vbeta8.2 between transgenic and wild-type mice. It is concluded that this microassay can serve as a quick, minimally invasive, non-lethal alternative for detecting TCR transgene-encoded antigens on the surface of mouse lymphocytes, and is potentially applicable to a number of other transgene-coded cell surface proteins.
Chou
Sonja T
ST
Department of Comparative Medicine, University of Washington School of Medicine, Seattle, Washington, USA.
Ladiges
Warren C
WC
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002452:Cell Count; D002469:Cell Separation; D005260:Female; D005434:Flow Cytometry; D008198:Lymph Nodes / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000378:metabolism; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics*
D016693:Receptors, Antigen, T-Cell, alpha-beta