کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
11025431 | 1689992 | 2018 | 30 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Efficient transgene insertion in a pseudorabies virus vector by CRISPR/Cas9 and marker rescue-enforced recombination
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کلمات کلیدی
Cas9CRISPR associated protein 9WSLASFVMCMVHCMVNHEJgRNAFRTeGFPDSBGFPCRISPRFBSPRVHDRCRISPR/Cas9 - CRISPR / Cas9BAC - LACclustered regularly interspaced short palindromic repeats - به طور منظم تکرار پینگندرومی کوتاه مدت میان دو طرف تقسیم می شودguide RNA - راهنمای RNAfetal bovine serum - سرم جنین گاوHuman cytomegalovirus - سیتومگالوویروس انسانیMurine cytomegalovirus - سیتومگالوویروس موشdouble strand break - شکست دو رشتهnon-homologous end joining - عدم پیوستن انتهای غیر همولوگhomology directed repair - هماهنگی کارگردانی تعمیرPseudorabies virus - ویروس PseudorabiesAfrican swine fever virus - ویروس تبخال آفریقاییenhanced green fluorescent protein - پروتئین فلورسنت سبز افزایش یافته استbacterial artificial chromosome - کروموزوم مصنوعی باکتریاییGlycoprotein D - گلیکوپروتئین D
موضوعات مرتبط
علوم زیستی و بیوفناوری
ایمنی شناسی و میکروب شناسی
ویروس شناسی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
For development of vectored vaccines against porcine pathogens the genome of the pseudorabies virus vaccine strain Bartha (PrV-Ba) was previously cloned as an infectious bacterial artificial chromosome (BAC), containing the bacterial replicon and a reporter gene cassette encoding enhanced green fluorescent protein (EGFP) at the nonessential glycoprotein G locus. To facilitate substitution of this insertion, this BAC was now modified by deletion of the adjacent promoter and initiation codon of the essential glycoprotein D (gD) gene of PrV-Ba. Furthermore, rabbit kidney (RK13) cells stably expressing Cas9 nuclease and an EGFP gene-specific guide RNA were prepared to induce site specific cleavage of the BAC DNA. After co-transfection of these cells with the modified BAC and recombination plasmids containing expression cassettes for new transgenes flanked by PrV DNA sequences including the intact 5â²-end of the gD gene, >95% of the recombinants exhibited the desired gene substitutions, while no EGFP-expressing progeny virus was detectable. This approach was used for insertion and expression of the open reading frames E199L, CP204L (p30) and KP177R (p22) of African swine fever virus. The studies revealed that codon adaptation significantly enhanced expression of E199L, and that the chimeric CAG promoter increased transgene expression compared to cytomegalovirus immediate-early promoters.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Virological Methods - Volume 262, December 2018, Pages 38-47
Journal: Journal of Virological Methods - Volume 262, December 2018, Pages 38-47
نویسندگان
Alexandra Hübner, Günther M. Keil, Tonny Kabuuka, Thomas C. Mettenleiter, Walter Fuchs,