Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line

Abstract

We report the development of an advanced system for transfer and expression of exogenous genes in mammalian cells based on Moloney murine leukemia virus (Mo MuLV). Extensive deletion/mutagenesis analysis to identify cis-acting signals involved in virus transmission has led to the design of a family of novel, highly efficient retroviral vectors and a partner helper-free packaging cell line. The pBabe retroviral vector constructs transmit inserted genes at high titres and express them from the Mo MuLV Long Terminal Repeat (LTR). Each of these vectors has been constructed with one of four different dominantly acting selectable markers, allowing the growth of infected mammalian cells in the presence of G418, hygromycin B, bleomycin/phleomycin or puromycin, respectively. The high titre ecotropic helper free packaging cell line, omega E, was designed in conjunction with the pBabe vectors to reduce the risk of generation of wild type Mo MuLV via homologous recombination events. The omega E cell line was generated with separate gagpol and ecotropic env expression constructs with minimal sequence overlap and decreased sequence homology achieved by 'codon wobbling'. Homologous env coding sequences were deleted from the pBabe vectors without diminishing recombinant vector titre. Together, the pBabe vectors and omega E cell line should prove useful in experiments where highest frequencies of gene transfer, or concomitant expression of several different genes within a single cell are required with minimal risk of helper virus contamination.

Anti-tumour activity against B16-F10 melanoma with a GM-CSF secreting allogeneic tumour cell vaccine

“…Transfection of K1735-M2 and B16-F10 cell lines with the GM-CSF gene A GM-CSF retroviral vector was made by splicing a fragment from the murine GM-CSF cDNA vector pXM (Genetics Institute) into the plasmid pBabeneo 40 which already had the neomycin resistance gene. PA317 packaging cells were transfected with the resulting plasmid by calcium phosphate precipitation and then selected in G418 containing medium.…”

Analysis of Brca1-deficient mouse mammary glands reveals reciprocal regulation of Brca1 and c-kit

“…Full-length mouse c-kit complementary DNA was PCR amplified from mice complementary DNA using the following primers c-kit FOR: 5 0 -AGCCACCGCGATGAGAGGC-3 0 , REV: TGCTCAGGC ATCTTCGTGCAC using Phusion polymerase (Finnzymes Oy, Espoo, Finland) and cloned into pGEM-T easy (Promega, Madison, WI, USA), and then subcloned into pBABE puromycin (Morgenstern and Land, 1990), which was used to transfect BOSC-23 packaging cells to produce ecotropic retroviruses. Filtered viral supernatant plus 4 mg/ml polybrene was used to infect recipient, the murine HC11 cell line.…”

A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

“…The amplified product was then EcoRI/XhoI-digested and ligated into EcoRI-SalI sites of the retroviral vector pBABE-puro [25]. The Lys52 to alanine mutation in the ATP binding site of CKLiK 296 was generated using the QuikChange Site Directed Mutagenesis kit (Stratagene) with the following primers: forward 5'-GGAAGCTCTTCGCAGTGGCGTGCATCCCG-3' and reverse 5'-CGGGATGCACGCCACTGCGAAGAGCTTCC-3'.…”