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Electron microscopy of hDmc1 nucleoprotein complexes. (a) hDmc1 incubated...
Electron microscopy of hDmc1 nucleoprotein complexes. (a) hDmc1 incubated with ssDNA in the presence of ATP. A short hDmc1 helical nucleoprotein filament is shown traversing the micrograph. This is...
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(A) Human embryonic stem (HESC) cell culture on day of transfection. The...
(A) Human embryonic stem (HESC) cell culture on day of transfection. The culture should be composed of many small (8–32 cells) colonies. (B) Transient expression of CMV-EGFP in HESCs after 48 h to...
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Schematic illustration describing the methods for generating genetically...
Schematic illustration describing the methods for generating genetically modified HESCs by infection.
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Schematic illustration describing the methods for generating genetically...
Schematic illustration describing the methods for generating genetically modified HESCs by transfection.
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Introduction of a mutation through the knock-in strategy. The targeting...
Introduction of a mutation through the knock-in strategy. The targeting vector carries a mutation (missense, nonsense, or small deletion) in the targeted exon (exon c in this example).
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Knock-in of cDNA by homologous recombination. In this targeting construct,...
Knock-in of cDNA by homologous recombination. In this targeting construct, one of the homology arms includes the 3’ UTR region of the targeted gene. Homologous recombination results in insertion of...
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Generation of a conditional knock-out allele. The vector for conditional...
Generation of a conditional knock-out allele. The vector for conditional knock-out is designed so that loxP sites are inserted flanking exon c. The neo r gene can be flanked by FRT sequences so that...
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Gene inactivation by homologous recombination. a, b, c, and d represent...
Gene inactivation by homologous recombination. a, b, c, and d represent exons of the wild-type gene. A conventional targeting construct carries positive (neo r ) and negative (HSV-tk) selection genes....
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Validation of targeted gene disruption and subsequent Cre-mediated...
Validation of targeted gene disruption and subsequent Cre-mediated recombination. (A) Generation of a null cell line from target gene. 5′ and 3′ specific sequences of the target gene are cloned into...
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A strategy for Cre-loxP recycling of the Bsr selectable marker. (A) The loxP...
A strategy for Cre-loxP recycling of the Bsr selectable marker. (A) The loxP recombination site includes a 13 bp inverted repeat separated by a spacer sequence. (B) The Floxed-Bsr cassette was...
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Highly Proficient Gene Targeting by Homologous Recombination in the Human...
Highly Proficient Gene Targeting by Homologous Recombination in the Human Pre-B Cell Line Nalm-6
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Highly Proficient Gene Targeting by Homologous Recombination in the Human...
Highly Proficient Gene Targeting by Homologous Recombination in the Human Pre-B Cell Line Nalm-6
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Highly Proficient Gene Targeting by Homologous Recombination in the Human...
Highly Proficient Gene Targeting by Homologous Recombination in the Human Pre-B Cell Line Nalm-6
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Analysis of the human KU86 locus using the genome browser of the University...
Analysis of the human KU86 locus using the genome browser of the University of California at Santa Cruz (...
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Agarose gel electrophoresis of candidate plasmids. Note that all the...
Agarose gel electrophoresis of candidate plasmids. Note that all the plasmids are undigested and therefore circular. (A) Screening of 5′-entry clone candidates after BP recombination. Clone 17 appears...
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Heterozygous disruption of the human KU86 gene. (A) Scheme for KU86...
Heterozygous disruption of the human KU86 gene. (A) Scheme for KU86 disruption in Nalm-6 cells. The human KU86 gene consists of 21 exons, located on chromosome 2q35 ( http://genome.ucsc.edu ). The...
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Schematic representation of targeting vector construction. The method is...
Schematic representation of targeting vector construction. The method is based on the MultiSite Gateway system, and consists of three steps: (1) genomic PCR to amplify attB-flanked 5′- and 3′-arms,...
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Generation of knockout mice. After successful gene targeting in ES cells,...
Generation of knockout mice. After successful gene targeting in ES cells, which grow as three-dimensional clumps on mouse embryo fibroblasts (MEFs), the cells are injected into a host blastocyst,...
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Gene targeting by double selection technique. Two fragments of the gene of...
Gene targeting by double selection technique. Two fragments of the gene of interest (thick lines) are cloned into a plasmid vector (thin lines) together with a neomycin resistance (neo) and a Herpes...
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Carcinogens Induce Deletions In Vivo in Mice
Carcinogens Induce Deletions In Vivo in Mice
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Preparation of an RPE. A round incision is made at the upper corneo-scleral...
Preparation of an RPE. A round incision is made at the upper corneo-scleral border of the eye to allow removal of cornea and lens. Six to eight incisions are made in the eye-cup from the...
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Close-up of eye-spots on the RPE of a p un mouse.
Close-up of eye-spots on the RPE of a p un mouse.
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A fur-spot on the p un mouse. The upper p un mouse is a control, and the...
A fur-spot on the p un mouse. The upper p un mouse is a control, and the lower mouse shows a black spot on its back and side.
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The p un mutation and possible mechanisms of intrachromosomal recombination...
The p un mutation and possible mechanisms of intrachromosomal recombination resulting in deletion of the repeated exons 6–18. The deletion can occur by several homologous recombination pathways, such...
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Engineering a conditionally targeted locus. A conditional gene-targeting...
Engineering a conditionally targeted locus. A conditional gene-targeting construct contains a positive selection marker (for selection of homologous recombinants) flanked by two loxP sites and two...
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Conditional targeting relies on the introduction of recombinase recognition...
Conditional targeting relies on the introduction of recombinase recognition sequences into a gene locus in an “innocuous” region ( Fig.
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Gene-targeting and transgenic animal models. (A) Transgenic mice may be...
Gene-targeting and transgenic animal models. (A) Transgenic mice may be engineered to express novel gene sequences from ubiquitous, tissue-specific, or inducible promoters. Gene-targeted mice may be...
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Conditional gene targeting requires crossing between two separate mouse...
Conditional gene targeting requires crossing between two separate mouse strains, one carrying the conditional allele and another expressing tissue-specific Cre recombinase (A). In the double mutant...
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Injection of ESC into the blastocoel cavity of a mouse blastocyst.
Injection of ESC into the blastocoel cavity of a mouse blastocyst.
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Generating subtle mutations with gene targeting. (A) Cre mediated...
Generating subtle mutations with gene targeting. (A) Cre mediated recombination. Recombination between two loxP sites (triangles) with the same orientation causes excision of the intervening segment...
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Schematic representation of vector/target recombination events using the...
Schematic representation of vector/target recombination events using the positive-negative selection approach. (A) Random vector insertion. (B) Gene targeting via homologous recombination (HR). (C)...
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Schematic representation of gene targeting in ESC.
Schematic representation of gene targeting in ESC.
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Microinjection of DNA solution into one of the pronuclei of a mouse zygote.
Microinjection of DNA solution into one of the pronuclei of a mouse zygote.
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Genetic and Phenotypic Characteristics of Various Human TNF Transgenic Mouse...
Genetic and Phenotypic Characteristics of Various Human TNF Transgenic Mouse Lines Generated in Our Laboratory
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DNA substrates recommended for binding and annealing assays
DNA substrates recommended for binding and annealing assays
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Kinetics of Rad52-mediated strand annealing: (a) Representative raw data...
Kinetics of Rad52-mediated strand annealing: (a) Representative raw data from a typical annealing experiment. Fluorescence intensity of Cy3 (black) and Cy5 (gray) are recorded in kinetics mode every...
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Binding of Rad52 protein to oligonucleotides of different lengths. (a)...
Binding of Rad52 protein to oligonucleotides of different lengths. (a) Binding titrations with human Rad52 and 1 nM ssDNA of various lengths were performed and the FRET efficiency (E FRET) was...
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Rad52 binding to the RPA–ssDNA complex: (a) Raw data from a representative...
Rad52 binding to the RPA–ssDNA complex: (a) Raw data from a representative experiment. Fluorescence intensity of Cy3 (black) and Cy5 (gray) are recorded in kinetic mode every 1 s. Arrows above the...
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RPA–ssDNA binding experiment: (a) Raw data from a representative experiment....
RPA–ssDNA binding experiment: (a) Raw data from a representative experiment. Fluorescence intensity of Cy3 (black) and Cy5 (gray) are recorded in kinetic mode every 1 s. Arrows above the graph...
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The Rad52 oligomer forms a ring structure with ssDNA binding site spanning...
The Rad52 oligomer forms a ring structure with ssDNA binding site spanning along the perimeter: structure of the undecameric ring of the DNA binding domain of human Rad52 protein (coordinates were...
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The scalloped wing phenotype indicates deletion into sequences flanking P{w...
The scalloped wing phenotype indicates deletion into sequences flanking P{w a } during repair. (a) Wild-type wing structure in a female fly. (b) A mild scalloped-wing phenotype resulting from a small...
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Cross schemes for the P{w a } assay when testing the role of the DmBlm...
Cross schemes for the P{w a } assay when testing the role of the DmBlm helicase in double-strand gap repair. (a) Females that are homozygous for P{w a } and heterozygous for one allele of mus309 are...
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Diagrammatic representation of ends-out targeting. Transgenic donor shows a...
Diagrammatic representation of ends-out targeting. Transgenic donor shows a clone using P[w35] as a basis for targeting. Hatched boxes are I-SceI recognition sites, half-arrows are FRTs, arrowheads...
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Outcomes of the repair of a double-strand break (DSB) introduced by I-SceI....
Outcomes of the repair of a double-strand break (DSB) introduced by I-SceI. (A) The integrated pim1-Q vector. Open arrows indicate the positions of PCR primers that are used to distinguish between the...
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Schematic representations of the (A) pim1-Q and (B) Rb-O targeting vectors....
Schematic representations of the (A) pim1-Q and (B) Rb-O targeting vectors. (A) Gene targeting strategy at the pim1 locus. Expression of the promotorless hygromycin resistance relies on the endogenous...
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