TG101348 was synthesized from the Memorial Sloan-Kettering Cancer Center Natural Synthesis Core Facility. Tofacitinib was pur- chased from Selleck. 17-AAG was bought from Selleck. PU-H71 6-amino-8- -N- -9H-purine- 9-propanamine hydrate was synthesized through the Chiosis Laboratory. Stock aliquots were ready in DMSO, stored at twenty C, and diluted in appro- priate media ahead of use. Ba/F3 cells have been maintained in RPMI 1640 medium with 10% FCS and 500 pg/ml IL-3 or one ng/ml TSLP. Ba/F3 had been stably trans- duced with CRLF2, IL7R, EpoR, HA JAK2, and Jak2 with or without activat- ing mutations within the pseudokinase domain as indicated. The B-ALL cell lines MUTZ-5 and MHH-CALL4 cells had been obtained from Deutsche Sammlung von Mikroorganismen und Zellkul- turen and grown in RPMI 1640 with 20% FBS. Random mutagenesis screen of human JAK2 R683G.
We modified a previously described strategy to make a randomly mutagenized cDNA library of human JAK2 R683G. In selleck quick, JAK2 R683G was cloned into the retroviral expression vector pMSCVneoatt, which was constructed by inserting Reading Frame Cassette A in to the multicloning blog of pMSCVpuro working with the Gateway Vector Conversion System, as previously described. In separate aliquots, we mutagenized a total of 100 ng DNA by transformation and propagation in XL-1 Red competent Escherichia coli, according to the manufacturers suggestions. Plasmid DNA was isolated applying Nucleobond Xtra Midi kit. For retrovirus manufacturing, we co-transfected the mutagenized JAK2 R683G cDNA library and the retroviral packaging construct pEcoPack at a 1:1 ratio into 293T cells by using Lipofectamine 2000. After 48 h, we harvested the supernatant, passed it by a 0.
45- m filter, and transduced thirty á 106 IL-3 dependent Ba/F3 cells that stably express CRLF2puro/IL7R-GFP. Immediately after 1 d, we washed the cells and resuspended them in fresh IL-3 containing media substituted with puromycin one g/ml. Soon after Huperzine A an alternative day, we changed media to no IL-3 and added one mg/ml neomycin. Cells had been then plated onto 96- or 384-well plates during the presence or absence of one M BVB808. Clones that survived BVB808 treatment were expanded in fresh RPMI 1640 media during the presence of puromycin/ neomycin/BVB808 and also the absence of IL-3. We isolated genomic DNA utilizing QIAamp DNA Blood Mini kit and amplified cDNA in pMSCVpuroatt by using the following primers. PCR items were recloned into retroviral expression vector using Gateway BP/LR Cloning Procedure, along with the ability to confer BVB808 resistance was confirmed by transduction and variety in CRLF2puro/IL7R-GFP expressing Ba/F3.
cDNA inserts from resistant clones have been then PCR amplified and Sanger sequenced at the Dana Farber Cancer Institute Molecular Biology Core Amenities or the DF/HCC DNA Sequencing Facility. Site-directed mutagenesis was per- formed applying the QuikChange II XL site-directed mutagenesis kit.