[...]... heterologous DNA, usually a drug selection gene or a marker gene to analyze target gene expression Insertion vectors (1,2), which may offer increased gene- targeting frequencies at a given genetic locus compared to a replacement vector (12), can also be used to disrupt gene function by inserting heterologous DNA, but also allow for the introduction of more subtle genetic alterations such as point mutations... in the gene- Gene Targeting in ES Cells 23 Fig 2 Targeting “nonselectable” and “selectable” genes (A) The vector contains a lacZ or GFP reporter gene cassette including a polyadenylation signal (stippled box) fused in-frame with target gene coding sequences A marker gene (neo or hyg) expression cassette (striped box) is uses for positive selection of ES cells The arrow indicates that the marker gene. .. current strategies for generating specific mutations in ES cells by describing the utility of the two general types of gene targeting vectors, describing the systems for inducing conditional mutations, and examining the parameters for designing a gene targeting vector for the experimental application of gene targeting in ES cells 1.1 Sequence Replacement Vectors The majority of gene- targeted mutations... not only for the study of gene function in vivo, but also serving as model systems for human disease Gene targeting is a term that is used to describe the predetermined mutation of an endogenous gene that results from a homologous recombination event between a mutated version of the gene carried on a targeting vector and the endogenous genetic locus When performed in ES cells, gene- targeting is a powerful... gene under the control of the tetOminimal promoter For more specificity, a tissue-specific promoter can be used to drive the expression of the TetR–VP16 gene Moreover, there are Tet-Off and Tet-On variations for controlling tet-responsive gene expression, which depend on the binding of tetracycline to TetR-VP16 Gene Targeting in ES Cells 29 Fig 5 Inducing gene- targeted mutations with Cre/loxP To generate... express an EcR-IRES-RxR gene cassette, (2) a transgene consisting of the muristerone A-responsive promoter to express the Cre gene, and (3) a conditionally targeted locus with the loxP site flanking the endogenous sequences to be deleted 1.4 Designing a Gene- Targeting Vector One of the initial considerations in the design of a gene- targeting experiment is the type and location of the genetic mutation to... drug-resistance gene expression cassette to allow for the positive selection of ES cells The most commonly used selectable marker has been the neomycin phosphotransferase gene (neo)(1) which confers resistance to the neomycin analog, G418 The neo gene is normally used as part of a cassette driven by the phosphoglycerate kinase gene (PGK) promoter or the herpes simplex virus thymidine kinase gene (HSVtk)... fusion with coding sequences to disrupt the target gene to serve as a sensitive marker to visualize target gene expression in mice heterozygous or homozygous for the mutant locus The second element of the cassette is an expression competent marker gene for the positive selection of vector-recipient ES cells 1.1.1.2 TARGETING OF SELECTABLE GENES If the target gene is expressed in ES cells, it is possible... contains the neo gene lacking an ATG and polyadenylation sequences fused in-frame with the target gene coding sequences Following homologous recombination at the target locus, neo gene expression will be regulated by the cis-acting sequences of the endogenous target gene to produce a neo fusion protein that confers resistance to G418 It has recently become possible to easily monitor target gene expression... the past several years new technologies have been developed that allow for inducible gene expression in transgenic mice that can be combined with more conventional gene- targeting approaches to control the timing and tissue-specificity of the desired mutation From: Methods in Molecular Biology, vol 158: Gene Knockout Protocols Edited by: M J Tymms and I Kola © Humana Press Inc., Totowa, NJ 19 20 DeChiara . h0" alt="" Gene Knockouts 1 1 From: Methods in Molecular Biology, vol. 158: Gene Knockout Protocols Edited by: M. J. Tymms and I. Kola © Humana Press Inc., Totowa, NJ Overview Gene Knockouts Paul. focus on the Gene Knockouts 3 practical issues in generating gene knockout mice, nevertheless it is worth- while discussing some of the general principles we have learned from those generated so. of establishing gene function through gene targeting is that a mouse with a null mutation in a specific gene is generated and whatever phenotype was observed indicates where the gene function was