Cells (Lonza) were plated at a density of 1.5 ?10 6 cells/ml and
Cells (Lonza) were plated at a density of 1.5 ?10 6 cells/ml and cultivated overnight in a serum free StemSpan SFEM medium (StemCell Technologies) with or without a cytokine cocktail composed of 100 ng/ml recombinant human stem cell factor (rhSCF), 20 ng/ml thrombopoietin (rhTPO), 100 ng/ml rhFlt3-L and 20 ng/ml interleukin 6 (rhIL6) (all from Peprotech). 1.5 ?10 7 transduction PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26080418 units of MMTV(SIN) or HIV-1 vectors were added to the cells. After the transduction period (20 h), vector-containing medium was replaced with the original medium (with or without cytokines) and cells were cultured for five days. Then, the cells were resuspended and maintained in the cytokine-containing medium. UV microscopy and FACS analysis were performed two weeks after transduction. Murine bone marrow cells were flushed from the femurs and tibias of eight-weeks-old Balb/c mice and the lineage negative (Lin-) cells were isolated using Lineage Cell Depletion kit (Miltenyi Biotech), following the manufacturer’s instructions. The day before transduction, the Lin- cells were resuspended at a density of 1 ?106 cells/ ml in StemSpan SFEM medium with or without cytokines consisting of 100 ng/ml recombinant murine stem cell factor (rmSCF), 100 ng/ml thrombopoietin (rmTPO), 100 ng/ml rmFlt3-L and 20 ng/ml interleukin 3 (rmIL3) (all from PeproTech). The betaretroviral or lentiviral vectors concentrated to 1.8 ?107 TU/ml were added toKonstantoulas and Indik POR-8 site retrovirology 2014, 11:34 http://www.retrovirology.com/content/11/1/Page 11 ofcells and after 24 h the transduced cells were cultured in the original medium. After five days the original medium was replaced with the medium containing cytokine cocktail and cells were cultured for an additional PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27693494 10 days.Irradiation of cellsHEK 293 cells were seeded at a density of 2 ?105 cells/ well (6 well plate) 24 h before exposure to a single radiation dose of 50 Gy using a linear accelerator (Siemens Primus, Munich, Germany). The -irradiated cells were used for transductions as well as cell cycle analysis by flow cytometry 24 h after the irradiation.Construction of plasmidsThe packaging construct pCMgpRRE17, derived from multiple fragments, was assembled in a series of subcloning steps. pCMMTV molecular clone that was used as a base for construction of the packaging construct was described elsewhere [9]. Due to a delayed processing of the Gag-Pro-Pol polyprotein, the gag-pro-pol-coding region in the pCMMTV was replaced with the corresponding region from a Mtv-1/MMTV(C3H) hybrid molecular clone [6]. The Mtv-1/MMTV(C3H) fragment was amplified using the hybrid molecular clone as a template and primers 1321FHind 5-AAA AAA AAG CTT GCA ACA GTC CTA ACA TTC AC-3and 6980R 5-CTC CTC CGC TTC GGA GAT-3(HindIII in italics), digested with HindIII (second HindIII site is in pol) and cloned into the 9.1 kb HinIII-HindIII backbone from pCMMTV. The resulting chimera pCMMTV1/2 was digested with XcmI and RsrII and ligated with the 2.1 kb XcmI-RsrII fragment obtained from pCMVrem (a Rem expression plasmid constructed by inserting the rem ORF into pcDNA3). The intermediate, env-3LTR lacking, product (pCM1/2env) was then processed using MluI and BstEII to delete the packaging signal (pCM1/ 2env). The digested vector was linked to a PCR product amplified using primers CMVdelF 5-ACA ACA AGG CAA GGC TTG AC -3and CMV3endR 5-AAA AAG GTG ACC GCT AGC AAT ATC GAT AAG CCA GTA AG-3 (BstEII in italics; MluI is downstream of CMVdelF) and digested with the same restric.
