Amplification Using CHO Cell Expression Vectors

The ability to select for integration of plasmid DNA into the host chromosome allows the generation of stably transfected cell lines. With transfection of a selectable marker linked to a nonselectable target gene (or by cotransfection of the two unlinked genes), high‐level expression of the desired gene is obtained by selecting for amplification of the selectable marker. This unit presents two systems for gene amplification and expression. The first describes the dihydrofolate reductase (DHFR) selection system while the second is based on selection of the glutamine synthetase (GS) gene. The DHFR system is probably more widely used, and results in very high levels of amplification and expression; however, the DHFR amplification process is lengthy and may require several months to isolate and characterize a stable, amplified line. In contrast, the GS system typically requires only a single round of selection for amplification to achieve maximal expression levels.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152963/1/cpmb1623.pd

A rapid embryonic stem cell-based mouse model for B-cell lymphomas driven by Epstein-Barr virus protein LMP1

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) contributes to oncogenic human B-cell transformation. Mouse B cells conditionally expressing LMP1 are not predisposed to B-cell malignancies, as LMP1-expressing B cells are eliminated by T cells. However, mice with conditional B-cell LMP1 expression and genetic elimination of alpha/beta and gamma/delta T cells ("CLT" mice) die early in association with B-cell lymphoproliferation and lymphomagenesis. Generation of CLT mice involves in-breeding multiple independently segregating alleles. Thus, although introduction of additional activating or knockout mutations into the CLT model is desirable for further B-cell expansion and immunosurveillance studies, doing such experiments by germline breeding is time-consuming, expensive, and sometimes unfeasible. To generate a more tractable model, we generated clonal CLT embryonic stem (ES) cells from CLT embryos and injected them into RAG2-deficient blastocysts to generate chimeric mice, which, like germline CLT mice, harbor splenic CLT B cells and lack T cells. CLT chimeric mice generated by this RAG2-deficient blastocyst complementation ("RDBC") approach die rapidly in association with B-cell lymphoproliferation and lymphoma. Because CLT lymphomas routinely express the activation-induced cytidine deaminase (AID) antibody diversifier, we tested potential AID roles by eliminating the AID gene in CLT ES cells and testing them via RDBC. We found that CLT and AID-deficient CLT ES chimeras had indistinguishable phenotypes, showing that AID is not essential for LMP1-induced lymphomagenesis. Beyond expanding accessibility and utility of CLT mice as a cancer immunotherapy model, our studies provide a new approach for facilitating generation of genetically complex mouse cancer models

Essential role for DNA-PKcs in DNA double-strand break repair and apoptosis in ATM-deficient lymphocytes

The DNA double-strand break (DSB) repair protein DNA-PKcs and the signal transducer ATM are both activated by DNA breaks and phosphorylate similar substrates in vitro, yet appear to have distinct functions in vivo. Here, we show that ATM and DNA-PKcs have overlapping functions in lymphocytes. Ablation of both kinase activities in cells undergoing immunoglobulin class switch recombination leads to a compound defect in switching and a synergistic increase in chromosomal fragmentation, DNA insertions, and translocations due to aberrant processing of DSBs. These abnormalities are attributed to a compound deficiency in phosphorylation of key proteins required for DNA repair, class switching, and cell death. Notably, both kinases are required for normal levels of p53 phosphorylation in B and T cells and p53-dependent apoptosis. Our experiments reveal a DNA-PKcs-dependent pathway that regulates DNA repair and activation of p53 in the absence of ATM

Genomic organization of mouse and human Bruton's agammaglobulinemia tyrosine kinase (Btk) loci

Btk is a cytoplasmic protein tyrosine kinase (PTK) that has been directly implicated in the pathogenesis of X-linked agammaglobulinaemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. We have isolated phage and cosmid clones that allowed us to deduce the genomic structure of mouse and human Btk loci. The mouse and human genes are contained within genomic regions that span approximately 43.5 kb and 37.5 kb, respectively. Both loci contain 18 coding exons ranging between 55 and 560 bp in size with introns ranging in size from 164 bp to approximately 9 kb. The 5'-untranslated regions are encoded by single exons located approximately 9 kb upstream of the first coding exon. Exon 18 encodes for the last 23 carboxyl-terminal amino acids and the entire 3'-untranslated region. The location of intron/exon boundaries in the catalytic domains of the mouse and human Btk loci differs from that found in other described sub-families of intracellular PTKs, namely that of Src, Fes/Fer, Csk, and Abl/Arg. This observation is consistent with the classification of Btk together with the recently characterized kinases, Tec and Itk, into a separate sub-family of cytoplasmic PTKs. Putative transcription initiation sites in the mouse and human Btk loci have been determined by using the rapid amplification of cDNA ends assay. Similar to many other PTK specific genes, the putative Btk promoters lack obvious TATAA and CAAAT motifs. Putative initiator elements and potential binding sites for Ets (PEA-3), zeste, and PuF transcription factors are located within the 300 bp which are located upstream of the major transcription start site in both species. These sequences can mediate promoter activity when placed upstream of a promotorless chloramphenicol acetyl transferase reporter gene in an orientation-dependent manner. The present analysis will significantly facilitate the mutational analyses of patients with XLA and the further characterization of the function and regulation of the Btk molecule