Transgenic Mice(轉基因小鼠) - 顯微注射用DNA 載體構建

1、Construct Transgene Vectors for Pronuclear InjectionDaniel ChapterHistory Apllications & Efficiency Factors Design Transgene Vectors Transgene vectors History-The First Transgenic Mice Transgenic History-The Super Mouse History-The transgenic Livestock Pronuclear Injection Pronuclear microinject

2、ion is conceptually straightforward: inject a small volume of fluid containing your gene of interest into a pronucleus of a zygote, and then transfer zygotes to a foster mother.Applications of Tansgene Technology Based on Pronuclear Injectionthe first group encompasses transgenics used for the inves

3、tigation of geneexpression patterns and the function ofregulatory sequences;In this group gene regulatory sequences are used in combination with a reportergene (e.g., LacZ to determine expressionpatterns. Applications of tansgene vectors In this case gene coding sequences are expressed under the con

4、trol of either tissue specific regulatory sequences or ubiquitously active promoters.The second group comprises transgenics designed to gain more insight into gene function.New applicationsLoss of Funtion Gain of Funtion Parameter That Influence The Efficiency of Producing Transgenic Animals by Pron

5、uclear MicroinjectionDNA concentration Injection buffer Visualizing pronuclei Timing of injection Size of DNA Copy number Position Effect insulator Well designed vectors can improveexpression of transgene!Design of a Transgene-Containing Vector Selection of Promoters and Additional Regulatory Sequen

6、ces for Tissue-Specific ExpressionFigure 5 Tissue-specific expression ofmGSTK1 and rGSTK1Biochem. J. (2003 373 (559569(Printed in Great BritainNevertheless, the suitability of the sequences identified has to be proven in vivo. Thesimplest approach uses combinations of promoter/enhancer deletions wit

7、h the LacZ orfluorescent protein reporter genes (luciferase.Selection of Promoters and Additional Regulatory Sequences for Ubiquitous Expression Insulatorschicken -globin 5 hypersensitive site 4 Selection and Design of the Coding Sequence cDNA-based transgenes often function in vivo, but expression

8、levels are frequently low, and such transgenes are often silenced.Inclusion of only one generic intron in a transgene has been shown to augment transgene expression significantlyThis hybrid intron, consisting of an adenovirus splice donor and an immunoglobulin G splice acceptor Selection of the Poly

9、adenylation Cassette SV40T polyA Human growth hormone gene rabbit -globin sequences Three vectors frequently used as trangenic vectors in Cyagen pRP.Des2d and pRP.Des3d; pHsp68 lacZ; BAC ; Lentiviral shRNA vecotr; pRP.Des2d and pRP.Des3d 1pRP/Des(R4R24812bpattR4Cm RccdBattR2SV40 pAAm R TB pUC ori f1 ori 1pRP/Des(R4R34812bp attR4ccdB Cm R attR3SV40 pA Am R TB pUC orif1 o riGateway Technologypromoter Gene 1st Gene 2nd Destination Vector Expression VectorEF1A-hrGFPCMV-hrGFP pHsp68 lacZ vector Based on the transcriptional mechanism of Heat shock pro