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Abstract Background Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare. Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. Results The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet-assisted transfection led to significantly lower efficiencies than the FHD protocol. With PLAL-AuNPs_S1 and _S2 the PI% was significantly higher, yet no consistent effect of these NPs on cell proliferation was observed. The magnet-assisted protocols were least effective, but did result in the lowest cytotoxic effect. Conclusions This study demonstrated that transfection efficiency of DNA-expression-plasmids was significantly improved by the addition of AuNPs. In some combinations the respective cytotoxicity was increased depending on the type of the applied AuNPs and the transfected DNA construct. Consequently, our results indicate that for routine use of these AuNPs the specific nanoparticle formulation and DNA construct combination has to be considered.

Abstract Ultrashort pulsed laser ablation in liquids represents a powerful tool for the generation of pure gold nanoparticles (AuNPs) avoiding chemical precursors and thereby making them especially interesting for biomedical applications. However, because of their electron accepting properties, laser-generated AuNPs might affect biochemical properties of biomolecules, which often adsorb onto the nanoparticles. We investigated possible effects of such laser-generated AuNPs on biological functionality of DNA molecules. We tested four differently sized and positively charged AuNPs by incubating them with recombinant eGFP-C1-HMGB1 DNA expression plasmids that code for eGFP fusion proteins and contain the canine architectural transcription factor HMGB1. We were able to show that successfully transfected mammalian cells are still able to synthesize and process the fusion proteins. Our observations revealed that incubation of AuNP with the plasmid DNA encoding the recombinant canine HMGB1 neither prevented the mediated uptake of the vector through the plasma membrane in presence of a transfection reagent nor had any effect on the transport of the synthesized fusion proteins to the nuclei. Biological activity of the recombinant GFP-HMGB1 fusion protein appears to have not been affected either, as a strong characteristic protein accumulation in the nucleus could be observed. We also discovered that transfection efficiencies depend on the size of AuNP. In conclusion, our data indicate that laser-generated AuNPs present a good alternative to chemically synthesized nanoparticles for use in biomedical applications.

Abstract Background Prostate cancer is a frequent finding in man. In dogs, malignant disease of the prostate is also of clinical relevance, although it is a less common diagnosis. Even though there are numerous differences in origin and development of the disease, man and dog share many similarities in the pathological presentation. For this reason, the dog might be a useful animal model for prostate malignancies in man. Although prostate cancer is of great importance in veterinary medicine as well as in comparative medicine, there are only few cell lines available. Thus, it was the aim of the present study to determine whether the formerly established prostate carcinoma cell line CT1258 is a suitable tool for in vivo testing, and to distinguish the growth pattern of the induced tumours. Methods For characterisation of the in vivo behaviour of the in vitro established canine prostate carcinoma cell line CT1258, cells were inoculated in 19 NOD.CB17-PrkdcScid/J (in the following: NOD-Scid) mice, either subcutaneously or intraperitoneally. After sacrifice, the obtained specimens were examined histologically and compared to the pattern of the original tumour in the donor. Cytogenetic investigation was performed. Results The cell line CT 1258 not only showed to be highly tumourigenic after subcutaneous as well as intraperitoneal inoculation, but also mimicked the behaviour of the original tumour. Conclusion Tumours induced by inoculation of the cell line CT1258 resemble the situation in naturally occurring prostate carcinoma in the dog, and thus could be used as in vivo model for future studies.

Abstract Background The high mobility group A1 proteins (HMGA1a/HMGA1b) are highly conserved between mammalian species and widely described as participating in various cellular processes. By inducing DNA conformation changes the HMGA1 proteins indirectly influence the binding of various transcription factors and therefore effect the transcription regulation. In humans chromosomal aberrations affecting the HMGA1 gene locus on HSA 6p21 were described to be the cause for various benign mesenchymal tumours while high titres of HMGA1 proteins were shown to be associated with the neoplastic potential of various types of cancer. Interestingly, the absence of HMGA1 proteins was shown to cause insulin resistance and diabetes in humans and mice. Due to the various similarities in biology and presentation of human and canine cancers the dog has joined the common rodent animal model for therapeutic and preclinical studies. Accordingly, the canine genome was sequenced completely twice but unfortunately this could not solve the structure of canine HMGA1 gene. Results Herein we report the characterisation of the genomic structure of the canine HMGA1 gene consisting of 7 exons and 6 introns spanning in total 9524 bp, the in vivo localisation of the HMGA1 protein to the nucleus, and a chromosomal assignment of the gene by FISH to CFA12q11. Additionally, we evaluated a described canine HMGA1 exon 6 SNP in 55 Dachshunds. Conclusion The performed characterisations will make comparative analyses of aberrations affecting the human and canine gene and proteins possible, thereby providing a basis for revealing mechanisms involved in HMGA1 related pathogenesis in both species.

Abstract Background Chromosomal translocations affecting the chromosome 2p21 cluster in a 450 kb breakpoint region are frequently observed in human benign thyroid adenomas. THADA (thyroid adenoma associated) was identified as the affected gene within this breakpoint region. In contrast to man tumours of the thyroid gland of dogs (Canis lupus familiaris) constitute mainly as follicular cell carcinomas, with malignant thyroid tumours being more frequent than benign thyroid adenomas. In order to elucidate if the THADA gene is also a target of chromosomal rearrangements in thyroid adenomas of the dog we have physically mapped the canine THADA gene to canine chromosome 10. A PCR was established to screen a canine genome library for a BAC clone containing the gene sequence of canine THADA. Further PCR reactions were done using the identified BAC clone as a template in order to verify the corresponding PCR product by sequencing. Canine whole blood was incubated with colcemid in order to arrest the cultured cells in metaphases. The verified BAC DNA was digoxigenin labeled and used as a probe in fluorescence in situ hybridization (FISH). Ten well spread metaphases were examined indicating a signal on canine chromosome 10 on both chromatids. A detailed fine mapping was performed indicating the canine THADA gene locus on the q-arm of chromosome 10. Results The canine THADA gene locus was mapped on chromosome 10q25. Our mapping results obtained in this study following the previously described nomenclature for the canine karyotype. Conclusion We analysed whether the THADA gene locus is a hotspot of canine chromosomal rearrangements in canine neoplastic lesions of the thyroid and in addition might play a role as a candidate gene for a possible malignant transformation of canine thyroid adenomas. Although the available cytogenetic data of canine thyroid adenomas are still insufficient the chromosomal region to which the canine THADA has been mapped seems to be no hotspot of chromosomal aberrations seen in canine thyroid adenomas.

The detailed analysis of the mRNA and protein structures carried out to identify TNF-[alpha], IL-1[alpha], and IL-1[beta] mRNAs and proteins among known mammalians such as human, canine, murine, and rat is presented.

The cytokines interleukin-1 (IL-1α and IL-1β) and the tumor necrosis factor-α (TNF-α) both play a major role in the initiation and regulation of inflammation and immunity responses. Polymorphisms within the gene sequences of these cytokines IL-1 and TNF-α have been proposed to play an important role in the pathogenesis of certain diseases. Affecting nearly ever), organ, various diseases, including some cancers, are described to be associated with an increased level of IL-1 and TNF-α proteins, for example, solid tumors, hematologic malignancies, malignant histiocytosis, autoimmune disorders, Alzheimer's disease, Parkinson's disease, sepsis, and rheumatoid arthritis. Regarding genetic backgrounds and pathways, numerous canine diseases show close similarities to their human counterparts. As a genetic model, the dog could be used to unravel the genetic mechanisms, for example, in particular the predispositions, the development, and progression of cancer and metabolic diseases. The identity comparison of gene and protein sequences of different species could be used to elucidate the structure and function of the genes and proteins by identifying the evolutionary conserved regions and domains. Herein we analyzed in detail the mRNA and protein structures and identities of the present known mammalian (human, canine, murine, rat, ovine, equine, feline, porcine, and bovine) TNF-α, IL-1α, and IL-1β mRNAs and proteins. Additionally, based on the canine genome sequence, we derived in silico the complete mRNA structures of the IL-1α and IL-1β mRNAs. [ABSTRACT FROM AUTHOR]

Point mutations within ras proto-oncogenes, particularly within the mutational hot-spot codons 12, 13 and 61, are frequently detected in human malignancies and in different types of experimentally-induced tumours in animals. So far little is known about ras mutations in naturally occurring canine fibrosarcomas or K-ras mutations in canine melanomas. To elucidate whether ras mutations exist in these naturally occurring tumours in dogs, in the present study we screened 13 canine fibrosarcomas, 2 feline fibrosarcomas and 11 canine melanomas for point mutations, particularly within the mutational hot-spots, making this the first study to investigate a large number of canine fibrosarcomas. None of the samples showed a K- or N-ras hot spot mutation. Thus, our data strongly suggest that ras mutations at the hot-spot loci are very rare and do not play a major role in the pathogenesis of the spontaneously occurring canine tumours investigated.