[Identification of unknown cadavers--multidisciplinary approach].

The comparison of fingerprints, dentition, genetic profile, imaging data and anthropological characteristics constitute the scientific basis for identification of unknown cadavers. The complexity of the identification process stems from a variety of factors such as the circumstances surrounding the death, the preservation state of the cadaver, availability of identification data and their quality, which affect the feasibility of the identification. This article reviews the most common identification techniques, the various sources of data accessible to the forensic scientist and the strategic decisions through which the unidentified body and the missing person are integrated.

ER-alpha-cDNA as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones.

Within the large group of Estrogen Receptor alpha (ERα)-negative breast cancer patients, there is a subgroup carrying the phenotype ERα(-), PR(-), and Her2(-), named accordingly "Triple-Negative" (TN). Using cell lines derived from this TN group, we wished to establish cell clones, in which ERα is ectopically expressed, forming part of a synthetic lethality screening system. Initially, we generated cell transfectants expressing a mono-cistronic ERα transcription unit, adjacent to a separate dominant selectable marker transcription unit. However, the yield of ERα expressing colonies was rather low (5-12.5%), and only about half of these displayed stable ectopic ERα expression over time. Generation and maintenance of such cell clones under minimal exposure to the ERα ligand, did not improve yield or expression stability. Indeed, other groups have also reported grave difficulties in obtaining ectopic expression of ERα in ERα-deficient breast carcinoma cells. We therefore switched to transfecting these cell lines with pERα-IRES, a plasmid vector encoding a bicistronic translation mRNA template: ERα Open Reading Frame (ORF) being upstream followed by a dominant-positive selectable marker (hygro(R)) ORF, directed for translation from an Internal Ribosome Entry Site (IRES). Through usage of this bicistronic vector linkage system, it was possible to generate a very high yield of ERα expressing cell clones (50-100%). The stability over time of these clones was also somewhat improved, though variations between individual cell clones were evident. Our successful experience with ERα in this system may serve as a paradigm for other genes where ectopic expression meets similar hardships.

Replication and episomal maintenance of Epstein-Barr virus-based vectors in mouse embryonal fibroblasts enable synthetic lethality screens.

Recently, we demonstrated the establishment of chemical and genetic synthetic lethality screens in cultured human cells. Here, we report the establishment of this method in mouse embryonal fibroblasts (MEF). The method employs an immortalized mammalian cell line, deficient in a gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest and the use of a novel green fluorescent protein (GFP)-based double-label fluorescence system. The crucial part in this endeavor has been the identification of a DNA replicon that could stably replicate in MEFs while under selection for survival and gets spontaneously lost relatively fast in the absence of such a pressure. Here, we show for the first time that EBV-based replicons but not polyoma virus-based ones can replicate and be stably maintained in MEFs. In the chemical screen, selective pressure imposed by synthetic lethal drugs prevented the spontaneous loss of the GFP-marked episome, enabling drug identification. Retention or spontaneous loss over time of the episomal survival plasmid could be sensitively detected in a large-scale blind test in the presence or absence of synthetic lethal chemicals, respectively. Establishing the synthetic lethality screen should thus permit high throughput screening for chemicals, which are synthetically lethal with any mouse mutant/knockout gene of interest. Moreover, it forms the basis for a genetic synthetic lethality screen in MEFs, an important new tool for mouse functional genomics.