Similarly, if any miscommunication arises in obtaining fresh patient tissue from the OR, we believe it is worthwhile to implant the tissue at whatever time it is eventually received since this primary tissue is so limited, and it is a reasonable to suggest the primary tumor would retain its viability past 24 hours. Lastly, our work will increase the ability of laboratories to share PDXs since now researchers at one institution can harvest fresh tumor and ship it overnight to another lab without needing to go through the process of slow freezing the tumor in dimethyl sulfoxide or glycerol first. Overall, we hope that our work will open the doors a bit wider for PDX establishment and propagation in order to increase the overall benefit of this model system. Recombinant protein Monensin sodium salt expression is a frequent necessity for biochemical studies of proteins, which cannot be obtained in high amounts from their natural source. Among these are many membrane proteins. Extensive expression screening is a vital initial step in the study of membrane proteins, and this stage involves substantial work with recombinant DNA to create the NCX 466 necessary expression constructs. Standard techniques of molecular biology, however, become limiting when working with gene sequences that are unstable in Escherichia coli. This is especially encountered in the case of mammalian membrane proteins. Comprehensive studies that address the actual cloning, propagation, and manipulation of constructs containing these unstable DNA sequences are rare and often lack detailed descriptions of the difficulties and, more importantly, of the solutions. In the case of the human bile salt export pump, the cloning of the cDNA into an expression vector only succeeded after a tremendous amount of work. This process resulted in a plasmid with several point mutations in the coding sequence, six of which changed the sequence on the protein level. A general issue encountered during propagation and targeted mutagenesis of BSEP-containing plasmids is the loss of various parts of the BSEP cDNA sequence in E. coli. This loss of sequences has been observed for other proteins as well. The corresponding DNA sequences have generally been termed ����unstable���� or even ����toxic����, because the presence of the intact plasmid ultimately resulted in bacterial cell death. One approach to create plasmids containing these ����toxic���� DNA fragments is to assemble it by homologous recombination in Saccharomyces cerevisiae thereby circumventing E. coli. S. cerevisiae is able to recombine several overlapping fragments into one circular plasmid containing the desired cDNA.