Exemplary Laboratory Technology

Obtaining cellular RNA and DNA. Transfer of genomic DNA from human cancer or leukemic cells into NIH 3T3 cells (established from mouse embryo fibroblasts) resulted in the transformation of 3T3 cells into focus-forming cells. Such focus-forming 3T3 cells first yielded the ras family (Harvey-, Kirsten-, N-ras) oncogenes. Co-transfection of such inoculated 3T3 cells into nude mice increased the sensitivity of the assay. 

The 6 X 10 inocula of NIH 3T3 cells were seeded 24 h earlier in a 100-mm culture dish and fed with 10 mL Dulbecco/Eagle s medium. Into each culture dish 1.25 mL DNA-precipitate is to be added and incubated at 37 °C for 15-18 h. Thereafter the Dulbecco/Eagle s medium is changed with 5 % fetal calf semm added. Culture fluids are changed x2/wk. Foci of cultured cells are looked for in 2-3 weeks. Once focus-forming cells are isolated and secured, they are tested by Southern blots with the human Alu repetitive sequence probe BLUR-84 for hybridization. Repeated cycles of transfection remove those human DNA sequences 

Chromosomal localization of the axl gene is done by fluorescence in situ hybridization (FISH). The biotin-labeled axl probe is from a cosmid vector containing a 41-kb genomic fragment. The slides with human metaphase cells for chromosomal DNA to be denatured are immersed into a solution of 70 % formamide-x4 in saline sodium citrate buffer (SSC) atpH 7.0 (0.15 M NaCl 0.015 M sodium citrate) at 70 °C for 2 min. For the slides, the hybridization mixture kept at 75 °C consists of 50 %formamide,xl SSC, 10 % dextran sulfate pH 7.0, and 150 mL unlabeled sonicated total human genomic DNA add 50-100 ng labeled probe. The mixture is incubated at 37 °C for 10 min. The slides are washed in 50 % formamide and SSC x3, 5 min each at 40 °C and in SSC x3 at 40 °C. The detection reagent consists of x4 SSC-0.1 % Triton, and 1 % bovine semm albumin for 3 washes 3 min each at 40 °C. The slides are incubated with fluorescein isothiocyanate-conjugated avidin at 37 °C for 30 min. Metaphase cells are counterstained with 4,6-diamidino-2-phenylindole dihydrochloride 200 ng/mL in 2x SSC for 5 min at 

Assays show that shed and solubilized TAM (Tyro3, Axl, MER) receptors circulate in high levels in the blood of patients with active systemic lupus erythematosus (SLE). These high titers of solubilized receptors correlated directly with disease activity, especially with that of lupus nephritis of low Clq and high anti-DNA antibody levels. In contrast, solubilized sMER levels did not correlate 

The vitamin K-dependent cytoplasmic Gas6 growth arrest-specific protein 6, a ligand, and its receptor axl/AXL. 

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