Our previous studies have suggested that harboring a soluble coxsackie-adenovirus receptor-ligand (sCAR-ligand) fusion protein expression cassette in the viral genome may provide a universal method to redirect oncolytic adenoviruses to various membrane receptors on cancer cells resisting to serotype 5 adenovirus infection. We report here a novel oncolytic adenovirus vector redirected to CD47+ l...

Adenovirus holds great promise as a gene delivery system; it can hold large amounts of exogenous DNA and can be chemically and genetically modified to improve targeting to specific cells and tissues. A recombinant adenovirus construct expressing p53 is currently in clinical use as a cancer therapy in China. However, the use of adenovirus constructs in therapy is limited due to patients' strong ...

The mouse Mitf gene encodes a transcription factor that is regulated by serine phosphorylation and is critical for the development of melanin-containing pigment cells. To test the role of phosphorylation at a particular serine, S73 in exon 2 of Mitf, we used a standard targeting strategy in mouse embryonic stem cells to change the corresponding codon into one encoding an alanine. By chance, we ...

The phage lambda-derived Red recombination system is a powerful tool for making targeted genetic changes in Escherichia coli, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or BAC targets. However, despite the common use of this system, the detailed mechanism by which lambda Red mediates double-stranded DNA recombination ...

Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene knock-out-technology in a variety of species. However, in a number of situations knocking out or modifying a single gene is not sufficient, this is particularly true for genes belonging to a common family or for genes showing redundant functions. Lik...

ATP-binding cassette, subfamily B (ABCB) 6 is a homodimeric ATP-binding cassette (ABC) transporter present in the plasma membrane and in the intracellular organelles. The intracellular localization of ABCB6 has been a matter of debate, as it has been suggested to reside in the mitochondria and the endo-lysosomal system. Using a variety of imaging modalities, including confocal microscopy and EM...

Existing methods for site-directed plasmid mutagenesis are restrained by the small spectrum of modifications that can be introduced by mutagenic primers and the amplicon size limitations of in vitro DNA synthesis. As demonstrated here, the combined use of Red/ET recombination and unique restriction site elimination enables extensive manipulation regardless of plasmid size and DNA sequence eleme...

Bone morphogenetic protein 2 (BMP2) was originally found by its osteoinductive ability, and recent genetic analyses have revealed that it plays critical roles during early embryogenesis, cardiogenesis, decidualization as well as skeletogenesis. In the course of evaluation of the conditional allele for Bmp2, we found that the presence of a neo cassette, a selection marker needed for gene targeti...

We tested the hypotheses that hypoxic stimulation enhances growth potentials of canine lymphoma cells by activating hypoxia-inducible factor 1α (HIF-1α), and that the hypoxia-activated prodrug (TH-302) inhibits growth potentials in the cells. We investigated how hypoxic culture affects the growth rate, chemoresistance, and invasiveness of canine lymphoma cells and doxorubicin (DOX)-resistant ly...

Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a reco...