A vast amount of data suggests that homologous recombination in mammalian cells is relatively rare as compared to random integration, imposing the need for sophisticated selection protocols to enrich for cells in which homologous recombination has occurred. We here show that one of the key factors in efficient homologous recombination is the use of isogenic DNA to prepare the targeting vectors. Homologous recombination at the retinoblastoma susceptibility gene (Rb) in embryonic stem cells derived from mouse strain 129 was 20-fold more efficient with a 129-derived targeting construct than with a BALB/c-derived construct. The two constructs were identical, except for a number of base sequence divergences between 129 and BALB/c DNA, including base-pair substitutions, small deletions/insertions, and a polymorphic CA repeat. Transfection with an isogenic DNA construct, containing 17 kilobases of homology, yielded a targeting frequency of 78% (of a total of 20,000 drug-resistant colonies), without the use of an enrichment protocol for homologous recombination. This result indicates that, also in mammalian cells, homologous recombination rather than random integration can be the predominant event.
A pgk::hprt fusion as a selectable marker for targeting of genes in mouse embryonic stem cells: disruption of the T-cell receptor delta-chain-encoding gene
Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines
A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure
Consecutive inactivation of both alleles of the pim-1 proto-oncogene by homologous recombination in embryonic stem cells
The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants
Production of chimaeric mice containing embryonic stem (ES) cells carrying a homoeobox Hox 1.1 allele mutated by homologous recombination
Germ-line transmission of a planned alteration made in a hypoxanthine phosphoribosyltransferase gene by homologous recombination in embryonic stem cells
Structure and expression of the murine retinoblastoma gene and characterization of its encoded protein
Inactivating the beta 2-microglobulin locus in mouse embryonic stem cells by homologous recombination
Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologous recombination in embryonic stem cells
Targeted homologous recombination at the endogenous adenine phosphoribosyltransferase locus in Chinese hamster cells
Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells
Most classical Mus musculus domesticus laboratory mouse strains carry a Mus musculus musculus Y chromosome
Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes
Replacement-like recombination induced by an integration vector with a murine homology flank at the immunoglobulin heavy-chain locus in mouse and rat hybridoma cells
Mouse genetics in the 21st century: using gene targeting to create a cornucopia of mouse mutants possessing precise genetic modifications
Disruption of the G(i2) alpha locus in embryonic stem cells and mice: a modified hit and run strategy with detection by a PCR dependent on gap repair
PCR-based gene targeting of the inducible nitric oxide synthase (NOS2) locus in murine ES cells, a new and more cost-effective approach
Derivation and comparison of C57BL/6 embryonic stem cells to a widely used 129 embryonic stem cell line
Unsuccessful attempt at gene-editing by homologous recombination in the zebrafish germ line using the approach of "Rong and Golic".
Targeting of exogenous DNA into Trypanosoma brucei requires a high degree of homology between donor and target DNA
Three new developments in P1 cloning. Increased cloning efficiency, improved clone recovery, and a new P1 mouse library
Targeted disruption of GAP-43 in P19 embryonal carcinoma cells inhibits neuronal differentiation. As well as acquisition of the morphological phenotype
Reduced X-ray resistance and homologous recombination frequencies in a RAD54-/- mutant of the chicken DT40 cell line
An efficient homologous recombination vector pTV(I) contains a hot spot for increased recombinant protein expression in Chinese hamster ovary cells
Structure of the mouse osteoclastogenesis inhibitory factor (OCIF) gene and its expression in embryogenesis
Abrogation of microsatellite-instable tumors using a highly selective suicide gene/prodrug combination
A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond
Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4-ligase IV during chromosomal translocation formation
53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers
Biosynthesis of major histocompatibility complex molecules and generation of T cells in Ii TAP1 double-mutant mice
Targeted disruption of CD44 in MDAY-D2 lymphosarcoma cells has no effect on subcutaneous growth or metastatic capacity
Mouse ubiquitous mitochondrial creatine kinase: gene organization and consequences from inactivation in mouse embryonic stem cells
Knockout of 'metal-responsive transcription factor' MTF-1 in Drosophila by homologous recombination reveals its central role in heavy metal homeostasis
Adenovirus-mediated Cre deletion of floxed sequences in primary mouse cells is an efficient alternative for studies of gene deletion
Human centromeric alphoid domains are periodically homogenized so that they vary substantially between homologues. Mechanism and implications for centromere functioning
Efficient transfer of base changes from a vector to the rice genome by homologous recombination: involvement of heteroduplex formation and mismatch correction
MSH6- or PMS2-deficiency causes re-replication in DT40 B cells, but it has little effect on immunoglobulin gene conversion or on repair of AID-generated uracils
Targeted gene modification in mismatch-repair-deficient embryonic stem cells by single-stranded DNA oligonucleotides
Production of alpha 1,3-galactosyltransferase-knockout cloned pigs expressing human alpha 1,2-fucosylosyltransferase
Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations
Ablation of the retinoblastoma gene family deregulates G(1) control causing immortalization and increased cell turnover under growth-restricting conditions
Interdigitated deletion complexes on mouse chromosome 5 induced by irradiation of embryonic stem cells
Disruption of imprinting and aberrant embryo development in completely inbred embryonic stem cell-derived mice
Mechanisms of recombination between diverged sequences in wild-type and BLM-deficient mouse and human cells
Coupled homologous and nonhomologous repair of a double-strand break preserves genomic integrity in mammalian cells
Hybrid embryonic stem cell-derived tetraploid mice show apparently normal morphological, physiological, and neurological characteristics
Plasminogen activator inhibitor-1 gene-deficient mice. I. Generation by homologous recombination and characterization
Restoration of mismatch repair functions in human cell line Nalm-6, which has high efficiency for gene targeting
Direct cloning of isogenic murine DNA in yeast and relevance of isogenicity for targeting in embryonic stem cells
Functional analysis in mouse embryonic stem cells reveals wild-type activity for three MSH6 variants found in suspected Lynch syndrome patients
Heterozygous insertions alter crossover distribution but allow crossover interference in Caenorhabditis elegans
Multiple copies of a linear donor fragment released in situ from a vector improve the efficiency of zinc-finger nuclease-mediated genome editing
Gene targeting by linear duplex DNA frequently occurs by assimilation of a single strand that is subject to preferential mismatch correction
Heteroduplex rejection during single-strand annealing requires Sgs1 helicase and mismatch repair proteins Msh2 and Msh6 but not Pms1
Mice without phosphatidylcholine transfer protein have no defects in the secretion of phosphatidylcholine into bile or into lung airspaces
Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining
DNA polymerases nu and theta are required for efficient immunoglobulin V gene diversification in chicken
Screening of HaCaT clones for CCL20 gene knockout and preliminary exploration of gene-targeting vector transfection approaches in this cell line
Transient suppression of MLH1 allows effective single-nucleotide substitution by single-stranded DNA oligonucleotides
Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants
Microinjection of Cre recombinase protein into zygotes enables specific deletion of two eukaryotic selection cassettes and enhances the expression of a DsRed2 reporter gene in Ccr2/Ccr5 double-deficient mice
Efficient production of Fah-null heterozygote pigs by chimeric adeno-associated virus-mediated gene knockout and somatic cell nuclear transfer
Rapid validation of cancer genes in chimeras derived from established genetically engineered mouse models
Disruption of the csk gene, encoding a negative regulator of Src family tyrosine kinases, leads to neural tube defects and embryonic lethality in mice
Highly efficient selection of the stable clones expressing antibody-IL-2 fusion protein by a dicistronic expression vector containing a mutant neo gene
Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease
Modeling oncogenic translocations: distinct roles for double-strand break repair pathways in translocation formation in mammalian cells
Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?
Meiotic prophase arrest with failure of chromosome synapsis in mice deficient for Dmc1, a germline-specific RecA homolog
Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer
Optimization of the production of knock-in alleles by CRISPR/Cas9 microinjection into the mouse zygote
ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes
Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines
Degenerated pIX-IVa2 adenoviral vector sequences lowers reacquisition of the E1 genes during virus amplification in 293 cells
Adenosine-deaminase-deficient mice die perinatally and exhibit liver-cell degeneration, atelectasis and small intestinal cell death
Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers
HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions
Long targeting arms do not increase the efficiency of homologous recombination in the beta-globin locus of murine embryonic stem cells.
Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation
Nonhomologous end joining during restriction enzyme-mediated DNA integration in Saccharomyces cerevisiae
A gene-targeting approach for functional characterization of KIAA genes encoding extremely large proteins
Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR-Cas9 genome editing
Molecular characterization of conventional and new repeat-induced mutants of nit-3, the structural gene that encodes nitrate reductase in Neurospora crassa
Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair
Investigation of the molecular biology underlying the pronounced high gene targeting frequency at the Myh9 gene locus in mouse embryonic stem cells
Looking back at multidrug resistance (MDR) research and ten mistakes to be avoided when writing about ABC transporters in MDR.
Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage
Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.
Alzheimer's Disease: MS4A
Variants within the membrane-spanning 4-domains subfamily A (MS4A) gene cluster have recently been implicated in Alzheimer's disease in genome-wide association studies. Here is the latest research on Alzheimer's disease and MS4A.
Pediculosis pubis is a disease caused by a parasitic insect known as Pthirus pubis, which infests human pubic hair, as well as other areas with hair including eye lashes. Here is the latest research.
Rh isoimmunization is a potentially preventable condition that occasionally is associated with significant perinatal morbidity or mortality. Discover the latest research on Rh Isoimmunization here.
Genetic Screens in iPSC-derived Brain Cells
Genetic screening is a critical tool that can be employed to define and understand gene function and interaction. This feed focuses on genetic screens conducted using induced pluripotent stem cell (iPSC)-derived brain cells. It also follows CRISPR-Cas9 approaches to generating genetic mutants as a means of understanding the effect of genetics on phenotype.
This feed focuses on molecular models of enzyme evolution and new approaches (such as adaptive laboratory evolution) to metabolic engineering of microorganisms. Here is the latest research.
Chronic Fatigue Syndrome
Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.
Pharmacology of Proteinopathies
This feed focuses on the pharmacology of proteinopathies - diseases in which proteins abnormally aggregate (i.e. Alzheimer’s, Parkinson’s, etc.). Discover the latest research in this field with this feed.
Alignment-free Sequence Analysis Tools
Alignment-free sequence analyses have been applied to problems ranging from whole-genome phylogeny to the classification of protein families, identification of horizontally transferred genes, and detection of recombined sequences. Here is the latest research.