Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 X 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced. (C) 2010 Elsevier Inc. All rights reserved.

Bovine sperm cells viability during incubation with or without exogenous DNA

The aim of this study was to assess the effect of exogenous DNA and incubation time on the viability of bovine sperm. Sperm were incubated at a concentration of 5 x 10(6)/ml with or without plasmid pEYFP-NUC. Fluorescent probes, propidium iodide/Hoechst 33342, FITC-PSA and JC-1, were used to assess plasma membrane integrity (PMI), acrosome membrane integrity (AMI) and mitochondrial membrane potential (MMP) respectively at 0, 1, 2, 3 and 4 h of incubation. Exogenous DNA addition did not affect sperm viability; however, incubation time was related to sperm deterioration. Simultaneous assessment of PMI, AMI and MMP showed a reduction in the number of sperm with higher viability (integrity of plasma and acrosome membranes and high mitochondrial membrane potential) from 58.7% at 0 h to 7.5% after 4 h of incubation. Lower viability sperm (damaged plasma and acrosome membranes and low mitochondrial membrane potential) increased from 4.6% at 0 h to 25.99% after 4 h of incubation. When PMI, AMI and MMP were assessed separately we noticed a reduction in plasma and acrosome membrane integrity and mitochondrial membrane potential throughout the incubation period. Therefore, exogenous DNA addition does not affect sperm viability, but the viability is reduced by incubation time.

Investigating the role of apoptosis regulator EndoG on exogenous DNA uptake, stability, replication and recombination

Endonuclease G (EndoG) is a well conserved mitochondrial nuclease with dual lethal and vital roles in the cell. It non-specifically cleaves endogenous DNA following apoptosis induction, but is also active in non-apoptotic cells for mitochondrial DNA (mtDNA) replication and may also be important for replication, repair and recombination of genomic DNA. The aim of our study was to examine whether EndoG exerts similar activities on exogenous DNA substrates such as plasmid DNA (pDNA) and viral DNA vectors, considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA and nuclease activity in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four day time-course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus (a cationic liposome transfection reagent), targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances it may non-specifically cleave intracellular DNA regardless of its origin. To investigate possible effects of EndoG on viral DNA vectors, we constructed and evaluated AdsiEndoG, a first generation adenovirus (Ad5 ΔE1) vector encoding a shRNA directed against EndoG mRNA, along with appropriate Ad5 ΔE1 controls. Infection of HeLa cells with AdsiEndoG at a multiplicity of infection (MOI) of 10 p.f.u./cell resulted in an early cell proliferation defect, absent from cells infected at equivalent MOI with control Ad5 ΔE1 vectors. Replication of Ad5 ΔE1 DNA was detected for all vectors, but AdsiEndoG DNA accumulated to levels that were 50 fold higher than initially, four days after infection, compared to 14 fold for the next highest control Ad5 ΔE1 vector. Deregulation of the cell cycle by EndoG depletion, which is characterized by an accumulation of cells in the G2/M transition, is the most likely reason for the observed cell proliferation defect. The enhanced replication of AdsiEndoG is consistent with this conclusion, as Ad5 ΔE1 DNA replication is intimately related to cell cycling and prolongation or delay in G2/M greatly enhances this process. Furthermore, infection of HeLa with AdsiEndoG at MOI of 50 p.f.u./cell resulted in an almost complete disappearance of viable, adherent tumour cells from culture, whereas almost a third of the cells were still adherent after infection with control Ad5 ΔE1 vectors, relative to the non-infected control. Therefore, targeting of EndoG by RNAi is a viable strategy for improving the oncolytic properties of first generation adenovirus vectors. In addition, AdsiEndoG-mediated knockdown of EndoG reduced homologous recombination between pDNA substrates in HeLa cells. The effect was modest but, nevertheless demonstrated that the proposed role of EndoG in homologous recombination of cellular DNA also extends to exogenous DNA substrates.

Liposome-mediated transfection of fetal lung epithelial cells: DNA degradation and enhanced superoxide toxicity.

Cationic liposomes, 1:1 (mol/mol) 1,2-dioleoyldimethylammonium chloride-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, were used to transfect primary cultures of distal rat fetal lung epithelial cells with pCMV4-based plasmids. A DNA-to-lipid ratio of 1:10 to 1:15 (wt/wt) optimized DNA uptake over a 24-h exposure. At a fixed DNA-to-lipid ratio of 1:15, chloramphenicol acetyltransferase (CAT) reporter gene expression declined at lipid concentrations > 2.5 nmol/cm2 cell surface area, whereas DNA uptake remained concentration dependent. CAT expression peaked 48 h after removal of the liposome-DNA complex, declining thereafter. Reporter gene expression was increased, and supercoiled cDNA degradation was reduced by the addition of 0.2 mM nicotinamide and 10 microM chloroquine. Rat fetal lung epithelial cells transfected with two different expression cassettes had an increased susceptibility to superoxide-mediated cytotoxicity. This could be attributed to a nonspecific delivery of exogenous DNA or some other copurified factor. The DNA-dependent increase in superoxide-mediated cytotoxicity, but not basal levels of cytotoxicity, was inhibited by the addition of 0.2 mM nicotinamide and 10 microM chloroquine.

DNase 2 is the main DNA-degrading enzyme of the stratum corneum

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA.

Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots

Organization of transgenes in rice transformed through direct DNA transfer strongly suggests a two-phase integration mechanism. In the “preintegration” phase, transforming plasmid molecules (either intact or partial) are spliced together. This gives rise to rearranged transgenic sequences, which upon integration do not contain any interspersed plant genomic sequences. Subsequently, integration of transgenic DNA into the host genome is initiated. Our experiments suggest that the original site of integration acts as a hot spot, facilitating subsequent integration of successive transgenic molecules at the same locus. The resulting transgenic locus may have plant DNA separating the transgenic sequences. Our data indicate that transformation through direct DNA transfer, specifically particle bombardment, generally results in a single transgenic locus as a result of this two-phase integration mechanism. Transgenic plants generated through such processes may, therefore, be more amenable to breeding programs as the single transgenic locus will be easier to characterize genetically. Results from direct DNA transfer experiments suggest that in the absence of protein factors involved in exogenous DNA transfer through Agrobacterium, the qualitative and/or quantitative efficiency of transformation events is not compromised. Our results cast doubt on the role of Agrobacterium vir genes in the integration process.

Induction of microsatellite instability by oxidative DNA damage

Instability of repetitive sequences, both in intronic sequences and within coding regions, has been demonstrated to be a hallmark of genomic instability in human cancer. Understanding how these mutational events arise may provide an opportunity for prevention or early intervention in cancer development. To study the source of this instability, we have identified a region of the β-lactamase gene that is tolerant to the insertion of fragments of exogenous DNA as large as 1,614 bp with minimal loss of enzyme activity, as determined by antibiotic resistance. Fragments inserted out-of-frame render Escherichia coli sensitive to antibiotic, and compensatory frameshift mutations that restore the reading frame of β-lactamase can be selected on the basis of antibiotic resistance. We have utilized this site to insert a synthetic microsatellite sequence within the β-lactamase gene and selected for mutations yielding frameshifts. This assay provides for detection of one frameshift mutation in a background of 106 wild-type sequences. Mismatch repair deficiency increased the observed frameshift frequency ≈300-fold. Exposure of plasmid containing microsatellite sequences to hydrogen peroxide resulted in frameshift mutations that were localized exclusively to the microsatellite sequences, whereas DNA damage by UV or N-methyl-N′-nitro-N-nitrosoguanidine did not result in enhanced mutagenesis. We postulate that in tumor cells, endogenous production of oxygen free radicals may be a major factor in promoting instability of microsatellite sequences. This β-lactamase assay may provide a sensitive methodology for the detection and quantitation of mutations associated with the development of cancer.

Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes

The uptake and expression of extracellular DNA has been established as a mechanism for horizontal transfer of genes between bacterial species. Such transfer can support acquisition of advantageous elements, including determinants that affect the interactions between infectious organisms and their hosts. Here we show that erythrocyte-stage Plasmodium falciparum malaria parasites spontaneously take up DNA from the host cell cytoplasm into their nuclei. We have exploited this finding to produce levels of reporter expression in P.falciparum that are substantially improved over those obtained by electroporation protocols currently used to transfect malaria parasites. Parasites were transformed to a drug-resistant state when placed into cell culture with erythrocytes containing a plasmid encoding the human dihydrofolate reductase sequence. The findings reported here suggest that the malaria genome may be continually exposed to exogenous DNA from residual nuclear material in host erythrocytes.

Kinetics and mechanism of DNA uptake into the cell nucleus

Gene transfer to eukaryotic cells requires the uptake of exogenous DNA into the cell nucleus. Except during mitosis, molecular access to the nuclear interior is limited to passage through the nuclear pores. Here we demonstrate the nuclear uptake of extended linear DNA molecules by a combination of fluorescence microscopy and single-molecule manipulation techniques, using the latter to follow uptake kinetics of individual molecules in real time. The assays were carried out on nuclei reconstituted in vitro from extracts of Xenopus eggs, which provide both a complete complement of biochemical factors involved in nuclear protein import, and unobstructed access to the nuclear pores. We find that uptake of DNA is independent of ATP or GTP hydrolysis, but is blocked by wheat germ agglutinin. The kinetics are much slower than would be expected from hydrodynamic considerations. A fit of the data to a simple model suggests femto-Newton forces and a large friction relevant to the uptake process.

Ataxia telangiectasia mutated (ATM) inhibition transforms human mammary gland epithelial cells.

Carriers of mutations in the cell cycle checkpoint protein kinase ataxia telangiectasia mutated (ATM), which represent 1-2% of the general population, have an increased risk of breast cancer. However, experimental evidence that ATM deficiency contributes to human breast carcinogenesis is lacking. We report here that in MCF-10A and MCF-12A cells, which are well established normal human mammary gland epithelial cell models, partial or almost complete stable ATM silencing or pharmacological inhibition resulted in cellular transformation, genomic instability, and formation of dysplastic lesions in NOD/SCID mice. These effects did not require the activity of exogenous DNA-damaging agents and were preceded by an unsuspected and striking increase in cell proliferation also observed in primary human mammary gland epithelial cells. Increased proliferation correlated with a dramatic, transient, and proteasome-dependent reduction of p21(WAF1/CIP1) and p27(KIP1) protein levels, whereas little or no effect was observed on p21(WAF1/CIP1) or p27(KIP1) mRNAs. p21(WAF1/CIP1) silencing also increased MCF-10A cell proliferation, thus identifying p21(WAF1/CIP1) down-regulation as a mediator of the proliferative effect of ATM inhibition. Our findings provide the first experimental evidence that ATM is a human breast tumor suppressor. In addition, they mirror the sensitivity of ATM tumor suppressor function and unveil a new mechanism by which ATM might prevent human breast tumorigenesis, namely a direct inhibitory effect on the basal proliferation of normal mammary epithelial cells.

Biochemical characterization of FANCD2 and FANCM in the Fanconi Anaemia pathway

Abstract : The maintenance of genome stability is a challenge for all living organisms. DNA is regularly subjected to chemical alterations by both endogenous and exogenous DNA damaging agents. If left unrepaired, these lesions will create mutations or lead to chromosomal instability. DNA crosslinking agents probably bring about the most toxic lesions. By linking covalently the two strands of DNA, crosslinking agents will impede essential cellular processes such as replication and transcription. Cells from Fanconi anaemia patients are extremely sensitive to these agents. Fanconi anaemia (FA) is a rare chromosomal instability disorder that leads to developmental defects, pancytopenia and cancer susceptibility. FA is a genetically heterogeneous disease with thirteen complementation groups identified. Proteins encoded by the FA genes work together in the FA pathway. Eight of these proteins form the FA core complex (FANC-A, B, C,E, F, G, L and -M), whose integrity is required to monoubiquitinate FANCD2 and FANCI in response to DNA damage. The hypersensitivity of FA cells to crosslinking agents, which perturb the progression of replication forks, has led to the hypothesis that FA proteins play a crucial role in the response to replication stress. However, at the molecular level, the functions of the FA pathway remain largely unknown. Our efforts were first focused on the characterization of FANCD2, "the key effector of the FA pathway". Using different substrates, we found that in vitro, purified hFANCD2 preferentially binds single strand DNA and double strand DNA extremities. Concomitantly, FANCM was identified as a new component of the FA core complex. Moreover FANCM was shown to have specific branch migration activities and probably a role as a "landing platform" on DNA for the other components of the core complex. By using FANCM mutants carrying deletions within the internal domain, we investigated the role of FANCM as a DNA anchor protein for the core complex. We observed that indeed, a specific part of the internal domain of FANCM interacts with components of the core complex. Finally, in collaboration with Weidong Wang's lab we characterized two new components of the FA pathway: FAAP10 and FAAP16. As a heterodimer these two proteins show affinity for dsDNA, and anneal complementary oligonucleotides in vitro. Moreover these proteins can associate with FANCM via a part of its internal domain. We find that FANCM, FAAP 10 and FAAP 16 can co-exist on the branch point of replication and recombination intermediates, and that FAAP10 and FAAP16 stimulate replication fork reversal by FANCM. These results suggest that FANCM may function as a landing platform for the core complex. After loading on DNA, the core complex can activate FANCD2 through monoubiquitination leading to its recruitment to the site of damage. Since ssDNA and double strand breaks are intermediates that are generated as a consequence of collapsed replication forks, FANCD2 by binding to ds DNA ends and ssDNA could protect such structures from the recombination repair machinery and prevent unscheduled recombination events. Alternatively, FANCD2 could avoid nucleases from gaining access to collapsed forks, preserving the DNA in state that can be used as a starting point for resumption of DNA synthesis. The overall comprehension of the FA pathway is far from been complete. Our results unravel new aspects of Fanconi Anaemia, which hopefully in the near future will address keys questions leading to a better understanding of the fascinating Fanconi Anaemia. Résumé : Le maintien de l'intégrité du génome est fondamentale chez tous les organismes vivants. L'ADN est constamment altéré par des composés aussi bien endogènes qu'exogènes. Si ces altérations ne sont pas réparées, elles peuvent conduire à l'apparition de mutations, ainsi qu'à une instabilité génomique accrue. Les lésions les plus sévères qui peuvent survenir sur l'ADN, sont les pontages inter caténaires. Des agents pontants en liant de façon covalente les deux brins d'ADN, vont empêcher le déroulement normal de processus cellulaires essentiels tels que la réplication ou la transcription. La compréhension des mécanismes permettant à la cellule de tolérer et réparer ces lésions est primordiale, notamment dans le cas des patients atteints de l'anémie de Fanconi qui présentent une très grande sensibilité à ces composés pontants. L'anémie de Fanconi est une maladie génétique rare appartenant à un groupe de pathologies associées à une grande instabilité chromosomique. Les patients atteints de l'anémie de Fanconi présentent des malformations du squelette, une pancytopénie et une forte propension à la survenue de cancer. L'anémie de Fanconi est génétiquement très hétérogène. À ce jour, 13 gènes codant pour 13 protéines FANC différentes ont été identifiés. Huit de ces protéines fonctionnent ensemble au sein d'un complexe (nommé le complexe FANC) ayant pour but de monoubiquitiner FANCD2 et FANCI en réponse à la formation de lésions sur l'ADN. L'extrême sensibilité des cellules de patients atteints de l'anémie de Fanconi à ces agents pontant l'ADN suggère l'implication des protéines FANC dans la réponse cellulaire suite à une stress réplicatif. Cependant, le rôle moléculaire exact de ces protéines demeure encore inconnu. Après purification, nous avons observé que FANCD2 était capable de lier l'ADN simple brin, ainsi que les extrémités d'ADN in vitro. Dans le même temps, FANCM fut identifié comme appartenant au complexe FANC. FANCM est décrit comme une translocase capable de promouvoir le déplacement de point de jonction dans des structures d'ADN spécifiques in vitro. De plus, en se liant à l'ADN, FANCM peut agir comme une plateforme pour les autres protéines FANC, leur permettant ainsi d'être adressées à l'ADN. En créant des protéines FANCM recombinantes ayant des délétions dans le domaine interne, nous avons pu observer que certaines protéines du complexe FANC se fixent à des sites spécifiques sur le domaine interne de FANCM. Enfin, au travers d'une collaboration, nous avons été amenés à caractériser deux nouvelles protéines appartenant au complexe FANC : FAAP 10 et FAAP16. Elles s'associent à FANCM par l'intermédiaire du domaine interne, et forment ainsi un hétérotrimére. La présence de FAAP10 et FAAP16 n'affecte pas la liaison de FANCM à l'ADN, mais semble potentialiser son activité de régression in vitro. FANCM semble donc fonctionner comme une plateforme pour les autres composants du complexe FANC. Ces derniers, une fois liés à l'ADN permettent la monoubiquitination de FANCD2 et son recrutement au site lésé de l'ADN. FANCD2 en se liant de façon préférentielle à l'ADN simple brin et aux extrémités d'ADN qui sont générés lors de l'arrêt et du démantèlement d'une fourche de réplication, pourrait protéger ces même fourches de réplication arrêtées, d'évènements de recombinaison aléatoires. Nos résultats apportent de nouveaux éléments concernant les mécanismes moléculaires de l'anémie de Fanconi. Enfin, l'étude de l'anémie de Fanconi permet aussi de mieux comprendre les mécanismes mis en place par la cellule pour tolérer des lésions survenant lors de la réplication.

Application of RNAi to silence tartrate-resistant acid phosphatase: unexpected effects on the monocyte-macrophage lineage

Tartraatti-resistentin happaman fosfataasin hiljentäminen RNAi menetelmällä: odottamaton vaikutus monosyytti-makrofagi linjan soluissa RNA interferenssi (RNAi) eli RNA:n hiljentyminen löydettiin ensimmäisenä kasveissa, ja 2000-luvulla RNAi menetelmä on otettu käyttöön myös nisäkässoluissa. RNAi on mekanismi, jossa lyhyet kaksi juosteiset RNA molekyylit eli siRNA:t sitoutuvat proteiinikompleksiin ja sitoutuvat komplementaarisesti proteiinia koodaavaan lähetti RNA:han katalysoiden lähetti RNA:n hajoamisen. Tällöin RNA:n koodaamaa proteiinia ei solussa tuoteta. Tässä työssä on RNA interferenssi menetelmän avuksi kehitetty uusi siRNA molekyylien suunnittelualgoritmi siRNA_profile, joka etsii lähetti RNA:sta geenin hiljentämiseen sopivia kohdealueita. Optimaalisesti suunnitellulla siRNA molekyylillä voi olla mahdollista saavuttaa pitkäaikainen geenin hiljeneminen ja spesifinen kohdeproteiinin määrän aleneminen solussa. Erilaiset kemialliset modifikaatiot, mm. 2´-Fluoro-modifikaatio, siRNA molekyylin riboosirenkaassa lisäsivät siRNA molekyylin stabiilisuutta veren plasmassa sekä siRNA molekyylin tehokkuutta. Nämä ovat tärkeitä siRNA molekyylien ominaisuuksia kun RNAi menetelmää sovelletaan lääketieteellisiin tarkoituksiin. Tartraatti-resistentti hapan fosfataasi (TRACP) on entsyymi, joka esiintyy luunsyöjäsoluissa eli osteoklasteissa, antigeenejä esittelevissä dendiriittisissä soluissa sekä eri kudosten makrofageissa, jotka ovat syöjäsoluja. TRACP entsyymin biologista tehtävää ei ole saatu selville, mutta oletetaan että TRACP entsyymin kyvyllä tuottaa reaktiivisia happiradikaaleja on tehtävä sekä luuta hajoittavissa osteoklasteissa sekä antigeenia esittelevissä dendriittisissä soluissa. Makrofageilla, jotka yliekpressoivat TRACP entsyymiä, on myös solunsisäinen reaktiivisten happiradikaalien tuotanto sekä bakteerin tappokyky lisääntynyt. TRACP-geenin hiljentämiseen tarkoitetut spesifiset DNA ja siRNA molekyylit aiheuttivat monosyytti-makrofagilinjan soluviljelymallissa TRACP entsyymin tuoton lisääntymistä odotusten vastaisesti. DNA ja RNA molekyylien vaikutusta TRACP entsyymin tuoton lisääntymiseen tutkittiin myös Tolllike reseptori 9 (TLR9) poistogeenisestä hiirestä eristetyissä monosyyttimakrofaagisoluissa. TRACP entsyymin tuoton lisääntyminen todettiin sekvenssistä ja TLR9:stä riippumattomaksi vasteeksi solun ulkopuolisia DNA ja RNA molekyylejä vastaan. Havainto TRACP entsyymin tuoton lisääntymisestä viittaa siihen, että TRACP entsyymillä on tehtävä solun immuunipuolustusjärjestelmässä.

A role for homologous recombination proteins in cell cycle regulation.

Eukaryotic cells respond to DNA breaks, especially double-stranded breaks (DSBs), by activating the DNA damage response (DDR), which encompasses DNA repair and cell cycle checkpoint signaling. The DNA damage signal is transmitted to the checkpoint machinery by a network of specialized DNA damage-recognizing and signal-transducing molecules. However, recent evidence suggests that DNA repair proteins themselves may also directly contribute to the checkpoint control. Here, we investigated the role of homologous recombination (HR) proteins in normal cell cycle regulation in the absence of exogenous DNA damage. For this purpose, we used Chinese Hamster Ovary (CHO) cells expressing the Fluorescent ubiquitination-based cell cycle indicators (Fucci). Systematic siRNA-mediated knockdown of HR genes in these cells demonstrated that the lack of several of these factors alters cell cycle distribution, albeit differentially. The knock-down of MDC1, Rad51 and Brca1 caused the cells to arrest in the G2 phase, suggesting that they may be required for the G2/M transition. In contrast, inhibition of the other HR factors, including several Rad51 paralogs and Rad50, led to the arrest in the G1/G0 phase. Moreover, reduced expression of Rad51B, Rad51C, CtIP and Rad50 induced entry into a quiescent G0-like phase. In conclusion, the lack of many HR factors may lead to cell cycle checkpoint activation, even in the absence of exogenous DNA damage, indicating that these proteins may play an essential role both in DNA repair and checkpoint signaling.

The establishment of two novel bovine cell lines by transfection with the putative transforming region of bovine adenovirus type 3

Human adenoviruses (Ads), members of the family adenoviridae, are medium-sized DNA viruses which have been used as valuable research tools for the study of RNA processing, oncogenic transformation, and for the development of viral vectors for use in gene delivery and immunization technology. The left 12% of the linear Ad genollle codes for products which are necessary for the efficient replication of the virus, as well as being responsible for the forlllation of tumors in animallllodels. The establishlllent of the 293 cell line, by immortalization of human embryonic kidney cells with th~ E1 region of Ad type S (AdS), has facilitated extensive manipulation of the Ads and the development of recombinant Ad vectors. The study of bovine adenoviruses (BAVs), which cause mild respiratory and gastrointestinal infections in cattle has, on the other hand, been limited primarily to that of infectivity, immunology and clinicallllanifestations. As a result, any potential as gene delivery vehicles has not yet been realized. Continued research into the molecular biolo~gy of BAVs and the development of recolllbinant vectors would benefit from the development of a cell line analogous to that of the 293 cells. In an attelllpt to establish such a cell line, the recombinant plaslllid pKC-neo was constructed, containing the left 0-19.7% of the BAV type 3 (BAV3) genome, and the selectable marker for resistance to the aminoglycoside G418, a neomycin derivative. The plasmid construct was then used to transfect both the Madin-Darby bovine kidney (MDBK) -iicell line and primary bovine lung cells, after which G418-resistant foci were selected for analysis. Two cell lines, E61 (MDBK) and E24 (primary lung), were subsequently selected and analysed for DNA content, revealing the presence of the pKC-neo sequences in their respective genomes. In addition, BAV3 RNA transcripts were detected in the E61 cells. Although the presence of E1 products has yet to be confirmed in both cell lines, the E24 cells exhibit a phenotype characteristic of partial transformation by E1. The apparent immortalization of the primary lung cells will permit exploitation of their ability to take up exogenous DNA at high efficiency.

Genetic Manipulation, Gene Silencing and Biomarker Development in Multiple Experimental Models.

The thesis is set in three different parts, according to the relative experimental models. First, the domestic pig (Sus scrofa) is part of the study on reproductive biotechnologies: the transgenesis technique of Sperm Mediated Gene Transfer is widely studied starting from the quality of the semen, through the study of multiple uptakes of exogenous DNA and lastly used in the production of multi-transgenic blastocysts. Finally we managed to couple the transgenesis pipeline with sperm sorting and therefore produced transgenic embryos of predetermined sex. In the second part of the thesis the attention is on the fruit fly (Drosophila melanogaster) and on its derived cell line: the S2 cells. The in vitro and in vivo models are used to develop and validate an efficient way to knock down the myc gene. First an efficient in vitro protocol is described, than we demonstrate how the decrease in myc transcript remarkably affects the ribosome biogenesis through the study of Polysome gradients, rRNA content and qPCR. In vivo we identified two optimal drivers for the conditional silencing of myc, once the flies are fed with RU486: the first one is throughout the whole body (Tubulin), while the second is a head fat body driver (S32). With these results we present a very efficient model to study the role of myc in multiple aspects of translation. In the third and last part, the focus is on human derived lung fibroblasts (hLF-1), mouse tail fibroblasts and mouse tissues. We developed an efficient assay to quantify the total protein content of the nucleus on a single cell level via fluorescence. We coupled the protocol with classical immunofluorescence so to have at the same time general and particular information, demonstrating that during senescence nuclear proteins increase by 1.8 fold either in human cells, mouse cells and mouse tissues.