Search results for "transgene"
showing 10 items of 259 documents
Transgene detection by digital droplet PCR
2014
Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 3…
Instability of Tandem Repetitive DNA in “Natural” and Transgenic Organisms
1996
Genome research of the last 10 years has forced us to re-evaluate our view of DNA as a relatively stable molecule. Unprecedented levels of DNA instability in germline and soma cells have been observed, associated primarily with tandem repetitive (tr) DNA sequences. We will discuss here briefly the structure and possible functions of trDNA in eukaryotes, the putative mechanisms of mutational change in repeat clusters and the evolutionary dimensions of trDNA instability (for other relevant reviews, sec Pardue and Hennig 1990; Vogt 1990; Charlesworth et al. 1994). A special focus will be on the behaviour of trDNA after DNA transfer experiments in transgenic organisms, with reference to our own…
Conditional transgenic mouse models: from the basics to genome-wide sets of knockouts and current studies of tissue regeneration
2008
Many mouse models are currently available, providing avenues to elucidate gene function and to recapitulate specific pathological conditions. To a large extent, successful translation of clinical evidence or analytical data into appropriate mouse models is possible through progress in transgenic or gene-targeting technology. Beginning with a review of standard mouse transgenics and conventional gene targeting, this article will move on to discussing the basics of conditional gene expression: the tetracycline (tet)-off and tet-on systems based on the transactivators tet-controlled transactivator (Tta) and reverse tet-on transactivator (rtTA) that allow downregulation or induction of gene exp…
Histone-mediated transgenerational epigenetics
2019
Abstract Epigenetic mechanisms operate at the interface between the environment and genome, by converting the environmental stimuli to phenotypic responses through changes in the chromatin landscape, which ultimately affects gene expression in the absence of alterations in DNA sequence. In this scenario, transgenerational inheritance occurs when epigenetic variations induced by environmental stimuli are transmitted through the germ line to succeeding generations that had never experienced those stimuli. There is an ever-growing list of reports indicating that histones are fundamental players in these processes in a variety of organisms. In this chapter, we provide a perspective on histone-d…
Transgenic tools for proteomic analysis of ciliary transport
2015
Vision begins as photons are captured by photoreceptor cilia and light is converted into electrical signals that are then sent to the brain. As the photoreceptor cilium is not able to make its own proteins, all polypeptides needed for converting photons into electrical signals are synthetized in the cell body. How these molecules move from the cell body to cilia is still unclear. Opsin is one of the best-characterized transmembrane proteins. Our goal is to understand the mechanism of opsin transport into photoreceptor cilia. In this project, we use a combination of genetic and proteomic approaches in the zebrafish model. As the first step, we are constructing a transgenic line that expresse…
A quick one-tube nested PCR-protocol for EPO transgene detection
2012
The practice of doping threatens fair competition in sports. With the very recent reports on successful gene therapies for several diseases, the likelihood for abuse of gene transfer techniques in elite sports is rapidly increasing. It is therefore very important to develop valid detection techniques for transgenic DNA (tDNA) with ultimate sensitivity and specificity. To date, three slightly different procedures have been reported to reliably detect tDNA with sufficiently high sensitivity. Two utilize a real-time PCR-based approach and one uses a primer-internal, intron-spanning PCR approach (spiPCR). The specificity and sensitivity of these techniques, however, is still a matter of debate.…
The expression level of GCNF affects fate choice during neural differentiation of PCC7 cells
2005
The nuclear receptor GCNF (NR6A1) is required for embryonic survival and development, and regulation of fertility. We used a transgenic approach to investigate its role in neural differentiation. As model we chose the embryonal carcinoma cell line PCC7, which reproducibly differentiates into a tissue-like pattern of neuronal and non-neuronal cells after exposure to retinoic acid (RA). The differentiation pattern of gcnf sense and antisense clones consistently indicated that the expression level of GCNF positively correlated with the development of the neuronal fate. Moreover, antisense clones failed to down-regulate expression of the key regulator of differentiation Oct4 during the initial …
Sleeping Beauty transposon system – future trend in T-cell-based gene therapies?
2006
Evaluation of: Huang X, Wilber AC, Bao L et al.: Stable gene transfer and expression in human primary T cells by the Sleeping Beauty transposon system. Blood 107, 483–491 (2006). The Sleeping Beauty (SB) transposon system can mediate stable gene transfer and expression in primary human T cells. Optimal in vitro conditions for maximum gene transfer efficiencies have been developed with regard to further application of the SB transposon system in T cell based gene therapies. This raises the question of whether or not the SB transposon system is a convincing alternative for virus-mediated gene transfer based on the currently available data. Here, we will discuss controversial safety and effic…
Gene silencing induced by oxidative DNA base damage: association with local decrease of histone H4 acetylation in the promoter region
2010
Oxidized DNA bases, particularly 7,8-dihydro-8-oxoguanine (8-oxoG), are endogenously generated in cells, being a cause of carcinogenic mutations and possibly interfering with gene expression. We found that expression of an oxidatively damaged plasmid DNA is impaired after delivery into human host cells not only due to decreased retention in the transfected cells, but also due to selective silencing of the damaged reporter gene. To test whether the gene silencing was associated with a specific change of the chromatin structure, we determined the levels of histone modifications related to transcriptional activation (acetylated histones H3 and H4) or repression (methylated K9 and K27 of the hi…
Virus replication and virion export in X-deficient hepatitis B virus transgenic mice
2002
The function of the X protein (pX) in the replication cycle of mammalian hepadnaviruses is enigmatic. Using tissue culture experiments it has been shown that the X gene product is not central to hepatitis B virus (HBV) replication and virion export. However, at present it is still unclear whether this also applies to the in vivo situation. Using a terminally redundant X-deficient HBV DNA construct, transgenic mice were established that exhibited high-level expression of the viral core protein in liver and kidneys. Importantly, replicative DNA intermediates and mature viral genomes could be detected in the liver and serum of these mice, respectively. These findings indicate that, in the in v…