Pwzl hygro 12s e1a

Timed heterozygous breeding between appropriate genotypes was used to generate embryos WT or null for HSF-1 or Hsp70. Retroviral particles were produced using the ecotropic Phoenix packaging cell line with the following plasmids: pWZL-hygro; pBABE-puro; pWZL-hygro 12S E1A or pBABE-puro K-Ras V12 (purchased from Addgene, Cambridge, MA). Primary WT or Hsp70^−/− MEFs were infected with virus for 6h before medium replacement, antibiotic selection and colony outgrowth visualized by methylene blue staining. In some experiments, transformed MEFs were maintained in culture for experimentation – the genotype was confirmed by qPCR and cell lines were maintained in vitro for a maximum of 10 passages before rescue of an earlier passage from liquid nitrogen storage. All cell lines generated were tested approximately every 3 months for mycoplasma contamination using MycoSEQ™ Mycoplasma Detection Assay (In Vitrogen) and discarded if found to be positive.