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Highly-efficient, fluorescent, locus directed cre and FlpO deleter mice on a pure C57BL/6N genetic background.
Birling MC, Dierich A, Jacquot S, Hérault Y, Pavlovic G
(2012) Genesis 50: 482-9
MeSH Terms: Animals, Bacterial Proteins, DNA Nucleotidyltransferases, Founder Effect, Gene Targeting, Genetic Engineering, Genotype, Green Fluorescent Proteins, Homologous Recombination, Integrases, Luminescent Proteins, Mice, Mice, Inbred C57BL, Mice, Transgenic, Proteins, RNA, Untranslated, Sequence Deletion
Show Abstract · Added March 20, 2013
To facilitate the use of the new mutant resource developed in the mouse, we have generated Cre and FlpO deleter mice on a pure inbred C57BL/6N background. The new transgenic constructs were designed to drive either the Cre or FlpO recombinase, fused to a specific fluorescent marker, respectively the eGFP or the eYFP, and were inserted by homologous recombination in the neutral Rosa26 locus. They allow a rapid, cost-effective, and efficient identification of the carrier individuals through the coexpression of the fluorescent marker. The recombination efficiency of the two deleter lines, Gt(ROSA)26S or < tm1(ACTB-cre,-EGFP)Ics> and Gt(ROSA) 26S or < tm2(CAG-flpo, EYFP)Ics>, was carefully evaluated using five loxP-flanked or four FRT-flanked alleles located at different positions in the mouse genome. For each tested locus, we observed a 100% excision rate. The transgenic mice are easily distinguishable from wild type animals by their bright fluorescence that remains easily detectable until 10 days after birth. In the adult, fluorescence can still be detected in the unpigmented paws. Furthermore, they both display accumulation of the specific recombinase during oogenesis. These fluorescent 'Cre- and Flp- deleter' transgenic lines are valuable tools for the scientific community by their high and stable recombination efficiency, the simplicity of genotype identification and the maintenance of a pure genetic background when used to remove specific selection cassette or to induce complete loss-of-function allele.
Copyright © 2012 Wiley Periodicals, Inc.
1 Communities
0 Members
0 Resources
17 MeSH Terms
A recombinase-mediated cassette exchange-derived cyan fluorescent protein reporter allele for Pdx1.
Potter LA, Choi E, Hipkens SB, Wright CV, Magnuson MA
(2012) Genesis 50: 384-92
MeSH Terms: Alleles, Animals, Cell Differentiation, Embryonic Stem Cells, Endoderm, Female, Flow Cytometry, Gene Expression Regulation, Developmental, Gene Regulatory Networks, Gene Targeting, Genes, Reporter, Green Fluorescent Proteins, Homeodomain Proteins, Mice, Mice, Inbred C57BL, Mice, Knockout, Microscopy, Fluorescence, Mutagenesis, Insertional, Pancreas, Recombinases, Trans-Activators
Show Abstract · Added December 5, 2013
Fluorescent protein (FP) reporter alleles are useful both for identifying and purifying specific cell populations in the mouse. Here, we report the generation of mouse embryonic stem cells that contain a pancreatic and duodenal homeobox 1 (Pdx1) loxed cassette acceptor (Pdx1(LCA)) allele and the use of recombinase-mediated cassette exchange to derive mice that contain a Pdx1(CFP) (Cerulean) reporter allele. Mice with this allele exhibited cyan fluorescence within the previously well-characterized Pdx1 expression domain in posterior foregut endoderm. Immunolabeling showed that endogenous Pdx1 was coexpressed with CFP at all time points examined. Furthermore, fluorescence-activated cell sorting was used to isolate CFP-positive cells from E11.5 and E18.5 embryonic tissues using both 405 and 445 nm lasers, although the latter resulted in a nearly 50-fold increase in emission intensity. The Pdx1(CFP) allele will enable the isolation of specific foregut endoderm and pancreatic cell populations, both alone and in combination with other FP reporter alleles.
Copyright © 2011 Wiley-Liss, Inc.
5 Communities
5 Members
5 Resources
21 MeSH Terms
Genetic mouse models for bone studies--strengths and limitations.
Elefteriou F, Yang X
(2011) Bone 49: 1242-54
MeSH Terms: Animals, Bone and Bones, Gene Deletion, Gene Targeting, Mice, Mice, Transgenic, Models, Animal, Promoter Regions, Genetic, Transgenes
Show Abstract · Added November 14, 2013
Mice have become a preferred model system for bone research because of their genetic and pathophysiological similarities to humans: a relatively short reproductive period, leading to relatively low cost of maintenance and the availability of the entire mouse genome sequence information. The success in producing the first transgenic mouse line that expressed rabbit β-globin protein in mouse erythrocytes three decades ago marked the beginning of the use of genetically engineered mice as model system to study human diseases. Soon afterward the development of cultured pluripotent embryonic stem cells provided the possibility of gene replacement or gene deletion in mice. These technologies have been critical to identify new genes involved in bone development, growth, remodeling, repair, and diseases, but like many other approaches, they have limitations. This review will introduce the approaches that allow the generation of transgenic mice and global or conditional (tissue-specific and inducible) mutant mice. A list of the various promoters used to achieve bone-specific gene deletion or overexpression is included. The limitations of these approaches are discussed, and general guidelines related to the analysis of genetic mouse models are provided.
Copyright © 2011 Elsevier Inc. All rights reserved.
0 Communities
2 Members
0 Resources
9 MeSH Terms
A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity.
Mussolino C, Morbitzer R, Lütge F, Dannemann N, Lahaye T, Cathomen T
(2011) Nucleic Acids Res 39: 9283-93
MeSH Terms: Bacterial Proteins, DNA Cleavage, DNA-Binding Proteins, Deoxyribonucleases, Gene Targeting, Genetic Engineering, Genome, Human, HEK293 Cells, Humans, Protein Engineering, Protein Structure, Tertiary, Trans-Activators, Transcription Activator-Like Effectors
Show Abstract · Added June 6, 2012
Sequence-specific nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger nucleases (ZFNs) and meganucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription activator-like effectors (TALEs) from the bacterial pathogen Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. In this study, we tested a panel of truncation variants based on the TALE protein AvrBs4 to identify TALE nucleases (TALENs) with high DNA cleavage activity. The most favorable parameters for efficient DNA cleavage were determined in vitro and in cellular reporter assays. TALENs were designed to disrupt an EGFP marker gene and the human loci CCR5 and IL2RG. Gene editing was achieved in up to 45% of transfected cells. A side-by-side comparison with ZFNs showed similar gene disruption activities by TALENs but significantly reduced nuclease-associated cytotoxicities. Moreover, the CCR5-specific TALEN revealed only minimal off-target activity at the CCR2 locus as compared to the corresponding ZFN, suggesting that the TALEN platform enables the design of nucleases with single-nucleotide specificity. The combination of high nuclease activity with reduced cytotoxicity and the simple design process marks TALENs as a key technology platform for targeted modifications of complex genomes.
1 Communities
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0 Resources
13 MeSH Terms
Genetic engineering of human pluripotent cells using TALE nucleases.
Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC, Zeitler B, Cherone JM, Meng X, Hinkley SJ, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R
(2011) Nat Biotechnol 29: 731-4
MeSH Terms: Base Sequence, Embryonic Stem Cells, Endonucleases, Gene Targeting, Genetic Engineering, Homeodomain Proteins, Humans, Induced Pluripotent Stem Cells, Molecular Sequence Data, Myosin-Light-Chain Phosphatase, Octamer Transcription Factor-3, Transcription Factors, Zinc Fingers
Show Abstract · Added June 6, 2012
Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).
1 Communities
0 Members
0 Resources
13 MeSH Terms
The flexible loop of Staphylococcus aureus IsdG is required for its degradation in the absence of heme.
Reniere ML, Haley KP, Skaar EP
(2011) Biochemistry 50: 6730-7
MeSH Terms: Bacterial Proteins, Binding Sites, Gene Targeting, Heme, Hemeproteins, Humans, Iron, Mixed Function Oxygenases, Mutant Chimeric Proteins, Oxygenases, Point Mutation, Protein Conformation, Protein Denaturation, Protein Stability, Staphylococcus aureus
Show Abstract · Added February 11, 2016
Degradation of specific native proteins allows bacteria to rapidly adapt to changing environments when the activity of those proteins is no longer required. Although these processes are vital to bacterial survival, relatively little is known regarding how bacterial proteins are recognized and targeted for degradation. Staphylococcus aureus is an important human pathogen that requires iron for growth and pathogenesis. In the vertebrate host, S. aureus fulfills its iron requirement by obtaining heme iron from host hemoproteins via IsdG- and IsdI-mediated heme degradation. IsdG and IsdI are structurally and mechanistically analogous but are differentially regulated by iron and heme availability. Specifically, IsdG is targeted for degradation in the absence of heme. Therefore, we utilized the differential regulation of IsdG and IsdI to investigate the mechanism of regulated proteolysis. In contrast to canonical protease recognition sequences, we show that IsdG is targeted for degradation by internally coded sequences. Specifically, a flexible loop near the heme-binding pocket is required for IsdG degradation in the absence of heme.
© 2011 American Chemical Society
0 Communities
1 Members
0 Resources
15 MeSH Terms
Targeted p120-catenin ablation disrupts dental enamel development.
Bartlett JD, Dobeck JM, Tye CE, Perez-Moreno M, Stokes N, Reynolds AB, Fuchs E, Skobe Z
(2010) PLoS One 5:
MeSH Terms: Ameloblasts, Animals, Cadherins, Catenins, Dental Enamel, Gene Expression Regulation, Developmental, Gene Targeting, Mice, Mice, Inbred C57BL, Mice, Knockout, Tooth
Show Abstract · Added March 28, 2014
Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows.
1 Communities
1 Members
0 Resources
11 MeSH Terms
NANOG reporter cell lines generated by gene targeting in human embryonic stem cells.
Fischer Y, Ganic E, Ameri J, Xian X, Johannesson M, Semb H
(2010) PLoS One 5:
MeSH Terms: Cell Line, Embryonic Stem Cells, Gene Expression Regulation, Gene Targeting, Genes, Reporter, Homeodomain Proteins, Humans, Nanog Homeobox Protein
Show Abstract · Added September 19, 2017
BACKGROUND - Pluripotency and self-renewal of human embryonic stem cells (hESCs) is mediated by a complex interplay between extra- and intracellular signaling pathways, which regulate the expression of pluripotency-specific transcription factors. The homeodomain transcription factor NANOG plays a central role in maintaining hESC pluripotency, but the precise role and regulation of NANOG are not well defined.
METHODOLOGY/PRINCIPAL FINDINGS - To facilitate the study of NANOG expression and regulation in viable hESC cultures, we generated fluorescent NANOG reporter cell lines by gene targeting in hESCs. In these reporter lines, the fluorescent reporter gene was co-expressed with endogenous NANOG and responded to experimental induction or repression of the NANOG promoter with appropriate changes in expression levels. Furthermore, NANOG reporter lines facilitated the separation of hESC populations based on NANOG expression levels and their subsequent characterization. Gene expression arrays on isolated hESC subpopulations revealed genes with differential expression in NANOG(high) and NANOG(low) hESCs, providing candidates for NANOG downstream targets hESCs.
CONCLUSION/SIGNIFICANCE - The newly derived NANOG reporter hESC lines present novel tools to visualize NANOG expression in viable hESCs. In future applications, these reporter lines can be used to elucidate the function and regulation of NANOG in pluripotent hESCs.
0 Communities
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1 Resources
8 MeSH Terms
Conditional gene targeting in mouse pancreatic ß-Cells: analysis of ectopic Cre transgene expression in the brain.
Wicksteed B, Brissova M, Yan W, Opland DM, Plank JL, Reinert RB, Dickson LM, Tamarina NA, Philipson LH, Shostak A, Bernal-Mizrachi E, Elghazi L, Roe MW, Labosky PA, Myers MG, Gannon M, Powers AC, Dempsey PJ
(2010) Diabetes 59: 3090-8
MeSH Terms: Animals, Brain, Crosses, Genetic, Estrogen Antagonists, Female, Galactosides, Gene Targeting, Genes, Reporter, Immunoglobulin G, Immunohistochemistry, Insulin, Insulin-Secreting Cells, Integrases, Leptin, Male, Mice, Mice, Transgenic, Reverse Transcriptase Polymerase Chain Reaction, Swine, Tamoxifen
Show Abstract · Added January 6, 2014
OBJECTIVE - Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell- or pancreas-specific recombination, also drive Cre expression in the brain.
RESEARCH DESIGN AND METHODS - Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1(tm1Cvw) lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry.
RESULTS - All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)(89.1Dam) mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)(1Lphi) mice were the only line that lacked Cre activity in the brain.
CONCLUSIONS - Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells.
3 Communities
5 Members
0 Resources
20 MeSH Terms
Multiple, temporal-specific roles for HNF6 in pancreatic endocrine and ductal differentiation.
Zhang H, Ables ET, Pope CF, Washington MK, Hipkens S, Means AL, Path G, Seufert J, Costa RH, Leiter AB, Magnuson MA, Gannon M
(2009) Mech Dev 126: 958-73
MeSH Terms: Animals, Basic Helix-Loop-Helix Transcription Factors, Body Patterning, Cell Differentiation, Cell Lineage, Cilia, Down-Regulation, Epithelium, Gene Expression Regulation, Developmental, Gene Silencing, Gene Targeting, Hepatocyte Nuclear Factor 6, Homeodomain Proteins, Insulin-Secreting Cells, Islets of Langerhans, Mice, Nerve Tissue Proteins, Pancreatic Ducts, Pancreatitis, Stem Cells, Time Factors, Tumor Suppressor Proteins
Show Abstract · Added January 6, 2014
Within the developing pancreas Hepatic Nuclear Factor 6 (HNF6) directly activates the pro-endocrine transcription factor, Ngn3. HNF6 and Ngn3 are each essential for endocrine differentiation and HNF6 is also required for embryonic duct development. Most HNF6(-/-) animals die as neonates, making it difficult to study later aspects of HNF6 function. Here, we describe, using conditional gene inactivation, that HNF6 has specific functions at different developmental stages in different pancreatic lineages. Loss of HNF6 from Ngn3-expressing cells (HNF6(Delta endo)) resulted in fewer multipotent progenitor cells entering the endocrine lineage, but had no effect on beta cell terminal differentiation. Early, pancreas-wide HNF6 inactivation (HNF6(Delta panc)) resulted in endocrine and ductal defects similar to those described for HNF6 global inactivation. However, all HNF6(Delta panc) animals survived to adulthood. HNF6(Delta panc) pancreata displayed increased ductal cell proliferation and metaplasia, as well as characteristics of pancreatitis, including up-regulation of CTGF, MMP7, and p8/Nupr1. Pancreatitis was most likely caused by defects in ductal primary cilia. In addition, expression of Prox1, a known regulator of pancreas development, was decreased in HNF6(Delta panc) pancreata. These data confirm that HNF6 has both early and late functions in the developing pancreas and is essential for maintenance of Ngn3 expression and proper pancreatic duct morphology.
4 Communities
5 Members
1 Resources
22 MeSH Terms