Optimized blood cell profiling method for genomic biomarker discovery using high-density microarray.

High-quality biomarkers for disease progression, drug efficacy and toxicity liability are essential for improving the efficiency of drug discovery and development. The identification of drug-activity biomarkers is often limited by access to and the quantity of target tissue. Peripheral blood has increasingly become an attractive alternative to tissue samples from organs as source for biomarker discovery, especially during early clinical studies. However, given the heterogeneous blood cell population, possible artifacts from ex vivo activations, and technical difficulties associated with overall performance of the assay, it is challenging to profile peripheral blood cells directly for biomarker discovery. In the present study, Applied BioSystems' blood collection system was evaluated for its ability to isolate RNA suitable for use on the Affymetrix microarray platform. Blood was collected in a TEMPUS tube and RNA extracted using an ABI-6100 semi-automated workstation. Using human and rat whole blood samples, it was demonstrated that the RNA isolated using this approach was stable, of high quality and was suitable for Affymetrix microarray applications. The microarray data were statistically analysed and compared with other blood protocols. Minimal haemoglobin interference with RNA labelling efficiency and chip hybridization was found using the TEMPUS tube and extraction method. The RNA quality, stability and ease of handling requirement make the TEMPUS tube protocol an attractive approach for expression profiling of whole blood to support target and biomarker discovery.

Will genetics really revolutionize the drug discovery process?

Genetics has played only a modest role in drug discovery, but new technologies will radically change this. Whole genome sequencing will identify new drug discovery targets, and emerging methods for the determination of gene function will increase the ability to select robust targets. Detection of single nucleotide polymorphisms and common polymorphisms will enhance the investigation of polygenic diseases and the use of genetics in drug development. Oligonucleotide arraying technologies will allow analysis of gene expression patterns in novel ways.

Analysis of GPT activity in mammalian cells with a chromosomally integrated shuttle vector containing altered gpt genes.

The molecular mechanisms of reversion in mammalian cells were studied utilizing the pZipGptNeo shuttle vector, with the bacterial gpt gene in the vector integrated into the chromosomal DNA of mouse cells. From mutant cell lines containing gpt genes with single base changes, revertants were selected for the reappearance of GPT activity. The copy number and expression of the gpt genes in such revertants were analyzed, and the GPT activity encoded by revertant genes in both mammalian cells and bacteria characterized. Revertants with wild-type amino acid sequence had, on average, the highest levels of GPT activity. Revertants with amino acid sequences different from the original mutants but not corresponding to wild-type had, on average, approximately half the level of GPT activity as wild-type revertants. Revertants that still contained the original mutation in the gpt gene had even lower levels of activity. These revertants were found to have amplified mutant gpt genes, which, when transferred into bacteria, were seen to encode for GPT polypeptides with partial enzymatic activity. A revertant in which the original mutation that destroyed the AUG translational start codon was retained but in which there was a secondary mutation upstream of the start codon also was characterized. The second mutation generated an in-frame CUG codon that apparently functioned as an alternative, upstream translational start codon.

A sensitive molecular assay for mutagenesis in mammalian cells: reversion analysis in cells with a mutant shuttle vector gene integrated into chromosomal DNA.

We have developed a system for the molecular analysis of mutations in mammalian cells. This system is based upon the use of mammalian cell lines containing mutant shuttle vector genes integrated into chromosomal DNA. The target for mutation was the Escherichia coli gpt gene, coding for the enzyme xanthine (guanine) phosphoribosyltransferase (GPT; EC 2.4.2.22). We have previously isolated a large number of cell lines containing mutant gpt genes with single base changes. From these lines, revertants were selected on the basis of the reappearance of GPT activity. In general, the frequency of revertants was below 10(-7). The gpt genes were recovered from 32 revertants and sequenced to determine the nature of the base changes associated with reversion. In the majority of the revertants, there was a base change within the originally mutated codon, leading to either restoration of the wild-type amino acid sequence or substitution of a different amino acid at the original mutated site. In no case did reversion of a base substitution mutant involve an amino acid residue other than that affected by the original mutation. The results have demonstrated a number of sites in the GPT polypeptide at which amino acid substitutions are compatible with enzyme activity and one site at which the loss of an amino acid is compatible with enzyme activity. This study establishes reversion analysis as a sensitive molecular assay for mutagenesis in mammalian cells.