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7) were electroporated with 20 μg of linearized DNA and subjected to positive selection with G418 (0.4 mg/ml) and negative selection with gancyclovir (1 μM). Two clones (designated 75 and 134) containing heterozygous disruptions of the gene were identified by Southern (DNA) blot analysis with probes flanking the 3′ homology region and with probes internal to both the 5′ and 3′ homology regions. The presence of a single integration event was verified by hybridization with a Neo probe (10). Germ line transmission was obtained with clone 75. For genotyping of mice, we subjected tail DNA to polymerase chain reaction analysis using two primer sets in the same reaction, one set (5′-TCCAAATGAAAAGAACGGCTATC-3′ and 5′-TGTACCAAAGTCACTGAAAAATCTGTT-3′) detecting the endogenous allele, and the other set (5′-AAGATAATATTGAAGCTGTAGGGAAAAA-3′ and 5′-TGTTAAGAAGGGTGAGAACAGAGTACC-3′) detecting a DNA segment in the targeted allele. Homozygous mutant cell lines were isolated by selecting heterozygous cells (clone 75) in increased concentrations of G418 (4.8 mg/ml). These lines and the parental clone 75 were subcloned before injection into RAG2-deficient blastocysts as described (11). Animal experiments were carried out in accordance with institutional guidelines.
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11
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0344243699
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32P-labeled DNA probes. Protein immunoblots were prepared with nitrocellulose membranes (Bio-Rad), probed with polyclonal or monoclonal antibodies, and developed with enhanced chemilumiscence reagents (NEN). Monoclonal antibody to p85α (anti-p85α) and polyclonal anti-pan-p85 were purchased from Upstate Biotechnology, Inc., polyctonal anti-p110α from Santa Cruz, and polyclonal anti-p85β was generated by immunizing rabbits with a keyhole limpet hemacyanin (KLH)-conjugated peptide derived from mouse p85β, TyrProPheArgArgGluArgProGluAspLeuGluLeu. Immunoprecipitations were performed with the same antibodies, except that the monoclonal anti-p85α was mixed with a second monoclonal anti-p85α from Transduction Laboratories, and the anti-p110α was a gift of K. Auger.
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Single-cell suspensions were prepared from lymphoid organs and red blood cells lysed in hypotonic buffer. Cells were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin- and CyChrome-labeled antibodies (Pharmingen and Southern Biotech) and analyzed on a FACScalibur with CellQuest software (Becton-Dickinson) with the use of a live cell gate.
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We measured Ig's in serial dilutions of serum using microtiter plates (Dynatech) and antibody pairs specific for different mouse Ig isotypes (Pharmingen). P-Nitrophenylphosphate was used as a substrate for the alkaline phosphatase-conjugated secondary antibodies, and the absorbance at 405 nm was measured in a microplate reader (Molecular Devices).
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3H]thymidine (20 μCi/ml, NEN) was added to each well. Cells were harvested 12 hours later with a TomTec plate harvester and counted in a BetaPlate scintillation counter (Wallac).
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We thank B. Sleckman, S. Thomas, G. Rathbun, and C. Carpenter for suggestions, K. Auger for anti-p110α, J. Lawitts for blastocyst injections, D. Pollard for mouse husbandry and genotyping, D. Kim for help with fibroblast culture, and K. Lee-Fruman for a critical reading of the manuscript. Supported by grants from NIH (to L.C.C., F.W.A., and S.B.S.), a fellowship from the Damon Runyon-Walter Winchell Cancer Research Fund (to D.A.F.), the Leukemia Society of America (to D.A.F.), and the Howard Hughes Medical Institute (to F.W.A.).
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