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    首頁> 外文學位 >Development of lentiviral vector mediated gene transfer techniques as tools to overexpress/knockdown P2X4 receptors in vitro.
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    Development of lentiviral vector mediated gene transfer techniques as tools to overexpress/knockdown P2X4 receptors in vitro.

    機譯:慢病毒載體介導的基因轉移技術的開發,作為在體外過表達/抑制P2X4受體的工具。

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    摘要

    The current study focused on the development of recombinant lentiviral (rLV) mediated gene transfer techniques that would aid in testing the hypothesis that the purinergic P2X4 receptors (P2X4Rs) play an important role in behavioral effects of alcohol. Ethanol inhibits ATP-induced currents in P2X4Rs recombinantly expressed in oocytes and HEK293 cells. Building evidence suggests that P2X4Rs play a role in alcohol consumption. P2X4Rs are widely expressed in neurons and glial cells, however little is known about the contribution of neuronal and/or glial P2X4Rs in alcohol-induced behaviors. In order to study the action of ethanol on P2X4Rs in these cells, it is necessary to develop tools to over-express/knockdown the expression of the receptors. The recombinant rLV gene delivery techniques were developed to express P2X4Rs in neuronal and HEK293 cells (Aim 1); to generate HEK293 cell line stably expressing P2X4Rs (Aim 2) and to inhibit P2X4Rs in microglial cells (Aim 3).;To address this issue, Lentivirus encoding P2X4R-GFP fusion construct was produced and was used to transduce neuronal and HEK293 cells. Fluorescence microscopy and western immunoblotting results confirmed the successful transduction of the P2X4Rs in the cells. The lentivirus encoding the P2X4R-GFP was then used to generate a HEK293 cell line stably expressing the P2X4Rs. Fluorescence microscopy and patch clamp technology was used to observe the stable expression of the P2X4Rs in HEK293 cells over a period of 47 days. A rLV containing shRNA oligonucleotides targeting the P2X4Rs was generated. Microglial BV-2 cells were transduced with the recombinant lentivirus and the knockdown of P2X4Rs was tested using fluorescence imaging and western immunoblotting. rLV infection of BV-2 cells with two different shRNA oligonucleotides resulted in more than 50% reduction of P2X4R protein expression per shRNA tested. Infection of BV2 cells with the combination of the two rLV shRNA oligonucleotides almost completely inhibited the expression of P2X4Rs (upto 85 %). The results suggest that the recombinant rLV vector can be successfully used for efficient gene delivery of P2X4Rs in neuronal and HEK293 cells as well as for knocking down P2X4Rs in microglial cells. This work sets the stage for the use of rLV technology to investigate the physiological and cellular roles of neuronal and microglial P2X4Rs in ethanol-induced behaviors.
    機譯:目前的研究集中于重組慢病毒(rLV)介導的基因轉移技術的發展,這將有助于檢驗嘌呤能P2X4受體(P2X4Rs)在酒精的行為效應中起重要作用的假設。乙醇抑制在卵母細胞和HEK293細胞中重組表達的P2X4Rs中ATP誘導的電流。有證據表明,P2X4Rs在飲酒中起作用。 P2X4Rs在神經元和神經膠質細胞中廣泛表達,但是對于神經元和/或神經膠質P2X4Rs在酒精誘導的行為中的貢獻知之甚少。為了研究乙醇對這些細胞中P2X4Rs的作用,有必要開發工具來過度表達/敲低受體的表達。開發了重組rLV基因遞送技術以在神經元和HEK293細胞中表達P2X4R(目的1);為了產生穩定表達P2X4R的HEK293細胞系(目的2)并抑制小膠質細胞中的P2X4R(目的3)。為了解決這個問題,生產了編碼P2X4R-GFP融合構建體的慢病毒并用于轉導神經元和HEK293細胞。熒光顯微鏡和免疫印跡結果證實了P2X4Rs在細胞中的成功轉導。然后將編碼P2X4R-GFP的慢病毒用于產生穩定表達P2X4Rs的HEK293細胞系。熒光顯微鏡和膜片鉗技術用于觀察HEK293細胞中P2X4R在47天內的穩定表達。產生了包含針對P2X4Rs的shRNA寡核苷酸的rLV。用重組慢病毒轉導小膠質細胞BV-2,并使用熒光成像和Western免疫印跡檢測P2X4Rs的敲低。用兩種不同的shRNA寡核苷酸對BV-2細胞進行rLV感染后,每個測試的shRNA導致P2X4R蛋白表達降低50%以上。用兩種rLV shRNA寡核苷酸的組合感染BV2細胞幾乎完全抑制了P2X4Rs的表達(高達85%)。結果表明重組rLV載體可以成功地用于神經元和HEK293細胞中P2X4Rs的有效基因傳遞以及敲除小膠質細胞中的P2X4Rs。這項工作為使用rLV技術研究神經元和小神經膠質P2X4R在乙醇誘導的行為中的生理和細胞作用奠定了基礎。

    著錄項

    • 作者

      Inamdar, Sneha.;

    • 作者單位

      University of Southern California.;

    • 授予單位 University of Southern California.;
    • 學科 Health Sciences Pharmacy.
    • 學位 M.S.
    • 年度 2010
    • 頁碼 64 p.
    • 總頁數 64
    • 原文格式 PDF
    • 正文語種 eng
    • 中圖分類
    • 關鍵詞

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