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Blood, Vol. 107, Issue 3, 1024-1030, February 1, 2006
Arrested natural killer cell development associated with transgene insertion into the Atf2 locus
Blood Kim et al.
107: 1024
Supplemental materials for: Kim et al, Vol 107, Issue 3, 1024-1030
Files in this Data Supplement:
- Figure S1. Fluorescent in situ hybridization (FISH) analysis of IL-2—activated NK cells (PDF, 23.4 KB) -
IL-2—activated NK cells from Tg mice (left) and wild-type (right) mice were produced under standard conditions.1 Cells were prepared for FISH by a modification of standard methods (see http://info.med.yale.edu/genetics/ward/tavi/fi01.html). Briefly, at day 5, the cells were treated with colcemid (10 ng/mL) for 5 hours, then treated with 0.56% KCl at 37°C for 5 minutes, and then fixed in solution containing methanol and acetic acid in a 3:1 ratio. The cells were stored at —20°C until use. The cells were then dropped onto noncoated microscope slides, dried, and dehydrated in ethanol. The slides were stored in 100% ethanol for 1 to 2 days, then treated for 2 minutes with pepsin (0.005%) in 0.01 N HCl at 37°C. The slides were then neutralized with PBS, dehydrated in ethanol, and air dried. The hybridization probe was the entire Ly49A cDNA from plasmid pA1.3 (Yokoyama et al2). The isolated insert was used for a biotin labeling reaction with the Bionick (Invitrogen, Carlsbad, CA) nick translation labeling system according to manufacturer’s directions. The labeled probe was purified with Qiaquick nucleotide removal kit according to manufacturer’s instructions (Qiagen, Valencia, CA). For each slide, 10 ng of the labeled probe was prepared with 40 µg Cot-1 DNA (Invitrogen) in Hybrisol 7 solution (Qbiogene, Irvine, CA). The probe was then added to the slides, coverslipped, and sealed with rubber cement. The slides were then placed on a ThermoBrite programmable slide processing instrument (Statspin, Norwood, MA). The slides were denatured at 75°C for 2 minutes and hybridized overnight at 37°C. The coverslip was then removed, and slides were washed 3 times with 50% formamide/2X SSC at 45°C for 5 minutes each, followed by 3 washes in 0.1X SSC at 60°C for 5 minutes each. The probe signal was amplified with a tyramide amplification kit containing strepavidin—horseradish peroxidase and FITC-tyramide (TSA Fluorescein System; Perkin Elmer, Boston, MA). The slides were then stained with 200 ng/mL DAPI in 4X SSC, washed, and mounted with Vectashield hardset mounting media (Vectorlabs, Burlingame, CA). The slides were visualized with a Nikon Eclipse E-400 equipped with filters for DAPI and FITC.
Note that the Ly49A cDNA used here constitutes a small probe for FISH that often produces only 1 or 2 signals per normal metaphase spread. Sometimes no signal is seen (see http://info.med.yale.edu/genetics/ward/tavi/fi16.html). There was always one very large bright signal on the spreads from the Tg NK cells, with smaller signals consistent with endogenous loci. No other signals were consistently seen on the Tg NK cell spreads. These results confirm a single transgene integration site with large copy number. Representative photomicrographs are shown. Magnification, 1000×. (The brightness of the Tg photomicrograph was diminished for the final print because of the brightness of the Tg insertion signal.)
References
1. Karlhofer FM, Ribaudo RK, Yokoyama WM. MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells. Nature. 1992;358:66-70.
2. Yokoyama WM, Jacobs LB, Kanagawa O, Shevach EM, Cohen DI. A murine T lymphocyte antigen belongs to a supergene family of type II integral membrane proteins. Journal of Immunology. 1989;143:1379-1386.
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