Zbornik - Društvo genetičara Srbije

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58 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE II-Pos-3 Subotica, 30. novembar - 4. decembar 2004. USPOSTAVLJANJE VEÆE EFIKASNOSTI TRANSFEKCIJE HUMANIH ÆELIJA U KULTURI Gordana Nikèeviæ, Nataša Kovaèeviæ-Grujièiæ, Isidora Petroviæ i Milena Stevanoviæ Institut za molekularnu genetiku and genetièko inenjerstvo, Beograd Transfer rekombinantnih gena u razlièite eukariotske æelije u kulturi je široko rasprostranjena metoda za prouèavanje genske ekspresije. Pošto je potreba za brzom, visoko efikasnom transfekcijom postala sve izraenija, razvijen je veliki broj produkata, ukljuèujuæi lipozome, nelipozomalne lipide, sintetièke polimere, i dr., koji omoguæavaju transport gena u æelije. Meðutim, problem kod veæine ovih, tzv. ne-viralnih agenasa je njihova niska efikasnost transfekcije. U cilju izuèavanja regulacije gena ukljuèenih u neuralnu diferencijaciju, bilo nam je neophodno da privremenom (transient) transfekcijom uvedemo rekombinantnu plazmidnu DNK u humanu embrionalno-karcinomsku æelijsku liniju (NT2/D1) sa visokom efikasnošæu. U tu svrhu, najpre smo testirali dva transfekciona sistema: LIPOFECTAMINE TM (Life Technologies) i EffecteneTM (QIAGEN) koristeæi pCH110 eukariotski vektor koji sadri lacZ reporter gen. Rezultati našeg istraivanja pokazali su da za NT2/D1 i HeLa æelije (model sistem koji se èesto koristi kao kontrola u eksperimentima transfekcije), efikasnost transfekcije EffecteneTM reagensom, moe jednostavno biti uveæana dodavanjem 1.5-3 puta veæe kolièine plazmidne DNK od one koja je preporuèena od strane proizvoðaèa. Koristeæi LIPOFECTAMINE TM reagens dobili smo optimalnu efikasnost transfekcije za obe æelijske linije sa preporuèenim koncentracijama, ali dodajuæi maksimalne kolièine plazmidne DNK. Zakljuèak našeg istraivanja je da prilikom optimizacije procesa transfekcije treba testirati i koncentracije plazmidne DNK znaèajno veæe od onih koje su preporuèene od strane proizvoðaèa u cilju postizanja visokog nivoa efikasnosti transfekcije humanih æelija u kulturi. IMPROVED TRANSFECTION EFFICIENCY OF CULTURED HUMAN CELLS The transfer of recombinant genes into variety of eukaryotic cultured cells is an extensively used approach in gene expression research. Since demand for rapid, high efficiency transfections become more intense, a number of products, including liposomes, non-liposomal lipids, synthetic polymers, etc., have been developed that mediate transport of genes into cells. However, problem associated with majority non-viral gene-delivery agents is their relatively low transfection efficiency. In order to study regulation of genes involved in neuronal differentiation, it was essential for us to transiently introduce recombinant plasmid DNA into NT2/D1 human embryonal carcinoma cell line with the high efficiency. For that purpose we first evaluated two transfection systems: LIPOFECTAMINE TM (Life Technologies) and EffecteneTM (QIAGEN) Transfection Reagents using pCH110 eukaryotic assay vector which contains functional lacZ reporter gene. We found out that under our culture conditions for NT2/D1 and HeLa cells (commonly used in transfections as a control), EffecteneTM transfection efficiency could simply be augmented by increasing the amount of plasmid DNA 1.5-3 times above recommended concentration without any visible cytotoxicity. With LIPOFECTAMINE TM reagent we obtained optimal transfection efficiency for both cell lines within the recommended concentrations, but with the highest point. Our conclusion is that optimization of transfection process should include plasmid DNA concentration above the level suggested by manufacturers in order to accomplish the highest transfection efficiency of human cells in culture.
II-Pos-4 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE 59 Subotica, 30. novembar - 4. decembar 2004. EKSPRESIJA I STRUKTURNA I FUNKCIONALNA ANALIZA PROMOTORA HUMANOG SOX18 GENA Isidora Petroviæ, Jelena Ðuroviæ, Gordana Nikèeviæ i Milena Stevanoviæ Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd Familija SOX gena sadri veliki broj gena koji se eksprimiraju tokom embriogeneze, a koji su u srodstvu sa sisarskim genom koji determiniše pol, oznaèenim kao SRY gen. Iako je samo nekoliko SOX gena detaljno analizirano, sve dosadašnje studije ukazuju na njihovu ulogu u regulaciji razviæa, a mutacije u nekim SOX genima su povezane sa pojavom oboljenja kod èoveka. Humani SOX18 gen je identifikovan pretraivanjem cDNK biblioteke napravljene iz mozga humanog fetusa, korišæenjem specifiène SOXA probe. Mapiranjem pozicije gena utvrðeno je da se SOX18 gen nalazi na hromozomu 20 u regionu q13,3. Pokazano je da mutacije u SOX18 genu dovode do pojave recesivne i dominantne forme sindroma koji obuhvata hipotrihozu, limfedem i teleangiektazije. Pored poznate funkcije u razvoju dlake i krvnih sudova, ovo ukazuje i na vanu ulogu SOX18 transkripcionog faktora u razvoju limfnih sudova. U cilju odreðivanja molekularnih mehanizama odgovornih za kontrolu ekspresije humanog SOX18 gena , analizirane su razlièite æelijske linije koje bi mogle da se koriste kao model sistem za buduæe eksperimente. Ekspresija SOX18 gena je analizirana u HUV-EC i EAhyb926 æelijskim linijama i detektovan je transkript duine 1,8 Kb. Karakterizacija SOX18 promotora obuhvatala je odreðivanje starta transkripcije metodom elongacije prajmera, kao i strukturne i funkcionalne analize regulatornog regiona odgovornog za ekspresiju SOX18 gena. Analizirana je sposobnost razlièitih fragmenata 5’ nekodirajuæeg regiona da utièu na ekspresiju cat reporter gena, a u cilju identifikacije DNK regiona odgovornog za ekspresiju SOX18 gena. EXPRESSION AND STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE PROMOTER OF HUMAN SOX18 GENE The SOX gene family consists of a large number of embryionically expressed genes that are related to the mammalian sex determining gene SRY. Although only few SOX genes have been characterized in detail, all studies indicate that these genes have important roles in the regulation of development and mutations in several SOX genes have been implicated in human diseases. Human SOX18 gene has been identified by screening human foetal cDNA library with specific SOXA probe. Mapping analysis has revealed that SOX18 gene is located on human chromosome 20q13.3. It has been shown that SOX18 mutations in human cause both recessive and dominant hypotrichosis- limphedema and telangiectasia, suggesting that, in addition to its established role in hair and blood vessel development, the SOX18 transcription factor plays a role in the development and/or maintenance of lymphatic vessels. In order to elucidate the molecular mechanisms controlling the expression of the human SOX18 gene, we have analyzed different cell lines that could be used as a model system for further experiments. The expression of SOX18 was studied by Northern blot analysis in HUV-EC and EAhyb926 cell lines and a single transcript of about 1.8 kb was detected. In order to characterize promoter of the SOX18 gene we have determined the transcription start site by primer extension analysis and carried out structural and functional analysis of the regulatory region responsible for SOX18 expression. To identify the DNA regions responsible for the control of the SOX18 transcription, we have analyzed the ability of truncated fragments from the 5’ flanking region to drive expression of cat reporter gene.
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DRUŠTVO GENETIČARA SRBIJE SERBIAN
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III KONGRES GENETIÈARA SRBIJE Subo
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ZAVRŠNO PREDAVANJE CLOSING LECTURE
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A Aksentijeviæ I. 194 Aleksiæ J.
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Zbornik - Društvo genetičara Srbije

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