Technology transfer from worms and flies to vertebrates: transposition-based genome manipulations and their future perspectives

To meet the increasing demand of linking sequence information to gene function in vertebrate models, genetic modifications must be introduced and their effects analyzed in an easy, controlled, and scalable manner. In the mouse, only about 10% (estimate) of all genes have been knocked out, despite continuous methodologic improvement and extensive effort. Moreover, a large proportion of inactivated genes exhibit no obvious phenotypic alterations. Thus, in order to facilitate analysis of gene function, new genetic tools and strategies are currently under development in these model organisms. Loss of function and gain of function mutagenesis screens based on transposable elements have numerous advantages because they can be applied in vivo and are therefore phenotype driven, and molecular analysis of the mutations is straightforward. At present, laboratory harnessing of transposable elements is more extensive in invertebrate models, mostly because of their earlier discovery in these organisms. Transposons have already been found to facilitate functional genetics research greatly in lower metazoan models, and have been applied most comprehensively in Drosophila. However, transposon based genetic strategies were recently established in vertebrates, and current progress in this field indicates that transposable elements will indeed serve as indispensable tools in the genetic toolkit for vertebrate models. In this review we provide an overview of transposon based genetic modification techniques used in higher and lower metazoan model organisms, and we highlight some of the important general considerations concerning genetic applications of transposon systems

Cell division promotes efficient retrotransposition in a stable L1 reporter cell line

Background: Long interspersed element type one (L1) actively modifies the human genome by inserting new copies of itself. This process, termed retrotransposition, requires the formation of an L1 ribonucleoprotein (RNP) complex, which must enter the nucleus before retrotransposition can proceed. Thus, the nuclear import of L1 RNP presents an opportunity for cells to regulate L1 retrotransposition post-translationally. The effect of cell division on L1 retrotransposition has been investigated by two previous studies, which observed varied degrees of inhibition in retrotransposition when primary cell strains or cancer cell lines were experimentally arrested in different stages of the cell cycle. However, seemingly divergent conclusions were reached. The role of cell division on retrotransposition remains highly debated. Findings: To monitor both L1 expression and retrotransposition quantitatively, we developed a stable dual-luciferase L1 reporter cell line, in which a bi-directional tetracycline-inducible promoter drives the expression of both a firefly luciferase-tagged L1 element and a Renilla luciferase, the latter indicative of the level of promoter induction. We observed an additional 10-fold reduction in retrotransposition in cell-cycle arrested cells even after retrotransposition had been normalized to Renilla luciferase or L1 ORF1 protein levels. In synchronized cells, cells undergoing two mitoses showed 2.6-fold higher retrotransposition than those undergoing one mitosis although L1 expression was induced for the same amount of time. Conclusions: Our data provide additional support for an important role of cell division in retrotransposition and argue that restricting the accessibility of L1 RNP to nuclear DNA could be a post-translational regulatory mechanism for retrotransposition

Transposon-based gene delivery vectors for gene therapy

The first gene therapy clinical trials were initiated more than two decades ago thanks to the previous development of viral vectors that allow high efficiency gene transfer into mammalian cells. Since then the application of viral gene transfer has been a successful treatment option for a variety of diseases. Hematopoietic stem cells (HSCs) represent the most frequently targeted cell population for the treatment of severe monogenic diseases as their gene therapeutic correction is a valid alternative to conventional HSC transplantation when a compatible donor is not available. Indeed, viral gene transfer was successfully applied in HSC-based ex vivo gene therapy of the blood and immune systems, albeit several studies have exposed serious adverse effects that were caused by the therapeutic vector induced inappropriate activation of proto-oncogenes. After these failures, researchers have developed new types of randomly integrating vectors that have proven safer in preclinical studies, which is consistent with interim reports of clinical trials also foreshadowing that they potentially have an improved safety profile. This review focuses on new and clinically relevant DNA transposon-based gene delivery vectors, and compares their properties with those of the old and new generation viral vectors

Rory Miller: Konfliktus és erőszak

Rory Miller tapasztalt harcművész és amerikai veterán nevelőtiszt nyugalmazott őrmesteri rendfokozatban. 1981 óta tanulmányozza a harcművészeteket, több száz könyvet elolvasott a témában. Könyveiben hangsúlyozza annak megértését, hogy a valódi önvédelmet és erőszakot a filmekből és a harcművészetekből származó mítoszok övezik, ami miatt az önvédelmet gyakran félreértik és hibásan tanítják. Könyvében megosztja ismereteit és tapasztalatait a börtönbeli verekedésekről, a taktikai műveletekről, rajtaütésekről, megmutatja a valódi erőszak arcát és összetettségét. Segít a harcművészeti és küzdősport képzési módszereket hozzáigazítani a valóságos küzdelem szabályaihoz

Gondolatok az intézkedéstaktikáról

Police tactics is a very important but neglected topic. There are just a few written documents about it, there is no schoolbook with comprehensive information about it, that’s why the education of police tactics is at low level. It’s probably related to the knowledge of the Hungarian police officers, which is not so perfect, I think. We need to collect all the knowledge we have about police tactics; we need to update it, we need to look for new information, and then we need to educate it at a higher level. My first step is determination of the concept police tactics.Az intézkedéstaktika egy igen jelentős, mégis nagyon elhanyagolt téma. Kevés róla az írásos anyag, a felgyülemlett tudásmennyiséget nem foglalja össze tankönyv, így az oktatása is gyenge lábakon áll. Ezzel feltehetően szoros összefüggésben van, hogy a rendőrök intézkedéstaktikai tudása – véleményem szerint – nem kielégítő. Szükség lenne a rendelkezésre álló tudásanyag összegyűjtésére, aktualizálására, új információk beszerzésére, majd megfelelő szinten történő oktatására. Kezdetnek az intézkedéstaktika fogalmának meghatározását tűztem ki célul

Rac1 Participates in Thermally Induced Alterations of the Cytoskeleton, Cell Morphology and Lipid Rafts, and Regulates the Expression of Heat Shock Proteins in B16F10 Melanoma Cells

Eukaryotic cells exhibit a characteristic response to hyperthermic treatments involving morphological and cytoskeletetal alterations and induction of heat shock protein synthesis. Small GTPases of the Ras superfamily are known to serve as molecular switches which mediate responses to extracellular stimuli. Here we addressed how small GTPase Rac1 integrates signals from heat stress and induces simultaneously various cellular changes in mammalian cells. Evidencing that Rac1 is implicated in the heat shock response, first we showed that both mild (41.5 °C) and severe (43 °C) heat shock induced membrane translocation of Rac1. Upon the inhibition of the activation (NSC23766) or palmitoylation (2-bromopalmitate) of Rac1, the size of its plasmamembrane bound pool was significantly decreased. Heat shock induced alterations in the cytoskeleton and cell morphology were prevented using the above inhibitors. Earlier we hypothesized that the level and size distribution pattern of Chol-rich rafts are directly coupled to the triggering mechanism of stress sensing and signaling. As a striking finding, when plasma membrane localization of Rac1 was inhibited we obtained reduced raft domain size at 43 °C. The above documented effects of Rac1 inhibitors were accompanied with a strongly decreased expression of hsp25 and hsp70 under both mild and severe heat stress conditions

Nemzetközi Rendészeti Figyelő VII.

Value: The international outlook draws readers' attention to the exciting topics of policing theory and law enforcement. It introduces the role of the human factor in forensic expert activity; we receive an assessment of international organized crime based on new criteria; we can study the world of assassinations; we meet video surveillance of public areas, and finally we talk about a specific area of ​​self-defense. Methodology: The methodologies draw attention to the human factors of expert errors, introduce economic aspects in the investigation of the causes of organized crime, recommend the so-called "scenario" method for analyzing criminality, present the effects of community supervision on populations with multiple disadvantages, in violent physical conflicts they call for an examination of mental reactions. Findings: Scientifically proven knowledge of individual law enforcement research topics can be more complete and useful for practice if researchers rely on methods that are well suited to the investigated phenomena. Value: The particular topics of law enforcement, examined with sufficient competence, can contribute to more effective protection of public safety.Cél: A nemzetközi kitekintés ezúttal is a rendészetelmélet és a bűnüldözés izgalmas témaköreire hívja fel az olvasók figyelmét. Megismertet az emberi tényező szerepével az igazságügyi szakértői tevékenységben; új szempontok alapján kapunk értékelést a nemzetközi szervezett bűnözésről; tanulmányozhatjuk a bérgyilkosságok világát; találkozunk a közterületek videós megfigyelésével, végül szó esik az önvédelem egy sajátos területéről.Módszertan: A metodikák felhívják a figyelmet a szakértői tévedések emberi tényezőire, közgazdasági szempontokat vezetnek be a szervezett bűnözés okainak vizsgálatánál, az úgynevezett „forgatókönyv” módszert javasolják a kriminalitás elemzéséhez, bemutatásra kerülnek a közösségi felügyelet hatásai a többszörösen hátrányos helyzetű populációkra, az erőszakos fizikai konfliktusokban a lelki reakciók vizsgálatát szorgalmazzák.Megállapítások: Az egyes rendészeti kutatási témák tudományosan is igazolt megismerése akkor lehet teljesebb és a gyakorlat számára is hasznosítható, ha a kutatók a vizsgált jelenségekhez jól illeszkedő módszerekre támaszkodnak.Érték: A rendészet partikuláris témakörei, kellő hozzáértéssel vizsgálva, hozzájárulhatnak a közbiztonság hatékonyabb védelméhez

Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers

<p>Abstract</p> <p>Background</p> <p>The transposon-based gene delivery technique is emerging as a method of choice for gene therapy. The <it>Sleeping Beauty </it>(SB) system has become one of the most favored methods, because of its efficiency and its random integration profile. Copy-number determination of the delivered transgene is a crucial task, but a universal method for measuring this is lacking. In this paper, we show that a real-time quantitative PCR-based, transgene-independent (qPCR-TI) method is able to determine SB transposon copy numbers regardless of the genetic cargo.</p> <p>Results</p> <p>We designed a specific PCR assay to amplify the left inverted repeat-direct repeat region of SB, and used it together with the single-copy control gene <it>RPPH1 </it>and a reference genomic DNA of known copy number. The qPCR-TI method allowed rapid and accurate determination of SB transposon copy numbers in various cell types, including human embryonic stem cells. We also found that this sensitive, rapid, highly reproducible and non-radioactive method is just as accurate and reliable as the widely used blotting techniques or the transposon display method. Because the assay is specific for the inverted repeat region of the transposon, it could be used in any system where the SB transposon is the genetic vehicle.</p> <p>Conclusions</p> <p>We have developed a transgene-independent method to determine copy numbers of transgenes delivered by the SB transposon system. The technique is based on a quantitative real-time PCR detection method, offering a sensitive, non-radioactive, rapid and accurate approach, which has a potential to be used for gene therapy.</p