bacterial artificial chromosome transgenic mice

One strategy to bypass these limitations is the use of bacterial artificial chromosome (BAC) transgenes. uptoThey are capable of carrying approximately 300 kbp of inserted DNA sequence. BAC Mice and Neuroscience Bacterial artificial chromosomes can contain genetic sequences that are known to be associated with conditions such as neurological disorders. ISBN 978-953-307-725-3, PDF ISBN 978-953-51-5175-3, Published 2011-11-25. . BACs produced transgenic lines that expressed the desired genes. . Generation of bacterial artificial chromosome (BAC) transgenic mice Transgenic mice are among the most helpful tools to study the role of genes in physiological conditions. A bacterial artificial chromosome ( BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid ), used for transforming and cloning in bacteria, usually E. coli. approach because the size of their inserts (ranging from This pioneer study triggered initial contradictory . Bacterial Artificial Chromosomes. congenic strain B6.D2-Mtv7a/Ty-27d were injected with RPCI-23 157J4 BAC DNA and three independent founder lines of BAC-transgenic . Super-Sized Inserts. Biotechnol. In this study we have generated and analyzed BAC transgenic mice carrying 168 kb of . . Swing and Shyam K. Sharan . Bacterial artificial chromosome (BAC) transgenes direct gene expression at physiological levels with the same developmental timing and expression patterns as endogenous genes in transgenic animal models. Bacterial artificial chromosome (BAC) transgenes direct gene expression at physiological levels with the same developmental timing and expression patterns as endogenous genes in transgenic animal models. BACs are composed of large (up to approximately 350 kb) pieces of genomic DNA. Transgenic mouse lines are characterized with Cre recombinase driven by promoters of CNS-specific genes using bacterial artificial chromosome (BAC) constructs. Segments of an organism's DNA, ranging from 100,000 to about 300,000 base pairs, can be inserted into BACs. Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a membrane localized cherry red fluorescent protein (mCherry), driven by the full-length SMA promoter and intronic sequences. Their stability and ease of handling have made these vectors increasingly popular for whole genome mapping and sequencing projects from microbes, plants, and animals. We created transgenic mice with a bacterial artificial chromosome (BAC) containing the human COL6A1 gene. Lu XH1, Fleming SM, Meurers B, Ackerson LC, Mortazavi F, Lo V, Hernandez D, Sulzer D, Jackson GR, Maidment NT, Bacterial artificial chromosome (BAC) transgenes direct gene. YEAST ARTIFICIAL CHROMOSOME (YAC) First described in 1983 by Murray and Szostak. Nkx2-5 BAC-GFP expression was copy number-dependent and locus site-independent. The F' plasmid allows bacteria to have "sex" (well, sort of: F' helps bacteria give . tion of bacterial artificial chromosome (BAC) transgenic mice that express enhanced green fluorescent protein (EGFP) specifically in glycinergic neurons under the con-trol of the GlyT2 promoter. The ability to transfer and maintain DNA both within and between species is an essential skill in biotechnology and medicine. PCR primer binding sites used in B and C are also indicated. In the first section, advantages of the BAC transgenic approach compared to the conventional transgenic approach are summarized. Human Bacterial Artificial Chromosome (BAC) Transgenesis Fully Rescues Noradrenergic Function in Dopamine beta-Hydroxylase Knockout Mice Joseph Cubells, Emory University Jason Schroeder, Emory University Elizabeth S. Barrie, Ohio State University Daniel F. Manvich, Emory University Wolfgang Sadee, Ohio State University Tiina Berg, Emory University Investigators have begun to employ these artificial chromosomes for the in vivo study of multigenic loci in mammalian cells. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. The α-syn BAC tg mice manifested decreased anxiety-like behaviors which may reflect non-motor symptoms of early PD, and they exhibited increased SERT . 15, 859-865. B. Shui. [1][2][3] F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. Over the lifetime, 2144 publication(s) have been published within this topic receiving 108225 citation(s). A powerful development is the bacterial artificial chromosome (BAC) transgenic approach, combining advantages of both conventional transgenic and knock‐in gene‐targeting strategies. The Transgenic Core guarantees that at least 50 blastocysts will be microinjected with ES cells to produce ES cell-mouse chimeras for the investigator. For example, a scientific study may identify a gene mutation that could make people more prone to seizures. Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. 26 Bacterial Artificial Chromosomes 4.1 Experimental design In our studies, we were interested in generating a reporter mouse line to monitor the expression of Smooth Muscle-D-Actin (SMA) in vivo and track the fate of cells expressing this gene. Nat. Transcriptional control of the SMA locus had been well defined in previous Enabling the bacteria to form a colony could provide a more long-term solution . To develop a new tool for the identification of in vitro enkephalinergic neurons and to analyze enkephalin promoter activity, we generated transgenic mice for a bacterial artificial chromosome (BAC). Thirty years after the first transgenic mouse was produced, a plethora of genetic tools has been developed to study immune cells in vivo.A powerful development is the bacterial artificial chromosome (BAC) transgenic approach, combining advantages of both conventional transgenic and knock‐in gene‐targeting strategies. They . A number of groups have used cosmid, ligated cosmid, or yeast artificial chromosome (YAC) constructs to try to recreate the native spatial architecture of the human β-globin locus in transgenic mice.1-8 These large constructs accurately recapitulate the normal cis environment required for high-level, tissue and developmental stage-specific . In mice and zebrafish, BAC-based reporters properly regulate gene expression irrespective of integration site when introduced randomly into the genome as a . In immunology the potential of BAC transgenic technology has yet . In this unit, detailed protocols are provided for modification (i.e., marker gene insertion, deletion, or point mutation) of BACs by homologous recombination in E. coli.This method utilizes a shuttle vector that allows transient expression of the E. coli RecA gene to support homologous . Since these regulatory sequences are often far apart, an ideal transgenic construct frequently needs to span more than 100 kb of DNA. Bacterial Artificial Chromosome Recombineering Core The mission of the BAC Core is to provide access to a complex technology to investigators at the so that they can use their resources to conduct research instead of developing tools for research. The present work presents a novel in vivo DRD1-Bacterial Artificial Chromosome (BAC) Tet-on system allowing for the inducible activation of tet-operated transgenes specifically within DRD1-expressing cells of transgenic mice. Recombineering of BAC DNA for the Generation of Transgenic Mice. Bacterial artificial chromosome A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. Michael Kotlikoff. Figure 1 Transgenic wild-type human PRSS2 and PRSS1 R122H expressions cooperatively initiated spontaneous pancreatitis. In the CNS of these mice, numerous EGFP-expressing neurons are found through-out the spinal cord, brainstem, and parts of the cerebel-lum. Bacterial Artificial Chromosome Recombineering: Recombineering is the process by which BACs are modified. Because the size of mammalian genes, including all of their associated regulatory elements, can exceed a megabase, transgenic complementation in mice has, in specific instances, proven to be a formidable hurdle. The transgenic mice carrying the different deletion mutations. 5. Two process can be used to obtain a sequenced genome, or region of . We here report a novel bacterial artificial chromosome (BAC) transgenic mouse line that expresses Class III β-tubulin fused to mCherry, an improved monomeric red fluorescent protein, for the visualization of microtubules during neuronal development. The resulting BAC transgenes can be used to produce transgenic mice, rats, or cell lines. Structure of hFOXP3 bacterial artificial chromosome (BAC) DNA. Bacterial artificial chromosome A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or cloning inF-plasmid), used for transforming and bacteria, usually E. Coli. Bacterial artificial chromosome is a(n) research topic. . The development of methods for engineering bacterial artificial chromosomes (BACs), and for the efficient production of BAC transgenic mice, has allowed the design of new in vivo approaches to the. If the bacteria do not form colonies inside the patient, the person must repeatedly ingest the modified bacteria in order to get the required doses. HC21, including the DSCR, were produced: (i) Ts65Dn mice In an effort to dissect the potential implication of SIM2 in the have three copies of MMU16 from App to Mx1 (20); and development of DS phenotypes, we have created two transgenic (ii) Ts1Cje mice have three copies of MMU16 from Sod1 to mouse lines using a bacterial artificial chromosome . To generate the Adipoq-Cre BAC transgenic mice, the 245 kb RP23-90G21 bacterial artificial chromosome (BAC) containing at least five mouse genes was modified by replacing the starting ATG and 222 bp of the adiponectin gene with the starting ATG and coding sequence of a Cre recombinase gene. "Homologous recombination based modification in Esherichia coli and germline transmission in transgenic mice of a bacterial artificial chromsome," Nature . A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. A (His) 6-Flag tag was inserted before the stop codon in exon 20 of Prestin.This tag is followed by the stop codon and an IRES/Cre cassette. Transgenic microbes have also been used in recent research to kill or hinder tumors, and to fight Crohn's disease. 15, 859-865. Transgenic mouse lines generated from this modified BAC clone closely resembled the endogenous Nkx2-5 expression in the heart, pylorus sphincter, and spleen, but expression was not detected in the tongue. It is shown that the DRD1-rtTA BAC-driven expression of a tet-operated reporter is under tight regulation by doxycycline . Bacterial Artificial Chromosomes (BAC) have been developed to hold much larger pieces of DNA than a plasmid can. Bacterial artificial chromosome vectors (BACs) were developed to permit the cloning and stable maintenance of large (100-200 kb) pieces of DNA in E. coli. Founder line 1 was analyzed. BAC vectors were originally created from part of an unusual plasmid present in some bacteria called the F' plasmid. the use of bacterial artificial chromosome (BAC) trans-genic mice for this purpose.The precise and efficient engineering of BACs can facilitate a variety of studies that cannot be readily accomplished using conventional approaches.I will discuss the molecular methods that are used to manipulate these large DNA constructs,the attB on the bacterial host. BACTERIAL ARTIFICIAL CHROMOSOME (BAC) TRANSGENESIS CONFIRMS EFFECT OF A QUANTITATIVE TRAIT LOCUS FOR SEIZURE SUSCEPTIBILITY IN MICE: FURTHER EVIDENCE THAT CODING VARIATION IN KCNJ10 INFLUENCES SEIZURE THRESHOLD . YACs are shuttle vectors capable of replicating and being selected in common bacterial hosts such as E.coli as well as in the yeast S. cerevisiae. Large fragments (50-200 kb) of the genomes of most animal model systems have been cloned into bacterial artificial chromosomes (BACs). In this protocol, we describe the generation of bacterial artificial chromosome (BACs) constructs, which are used to express a gene of interest under a particular promoter. we generated bacterial artificial chromosome-transgenic (BAC-Tg) mouse lines containing the human RCAN1 gene with a C-terminal HA-FLAG epitope . DA neurons in vivo, we developed a Bacterial Artificial Chromosome (BAC) transgenic mouse model expressing a C-terminal truncated human mutant parkin (Parkin-Q311X) in DA neurons driven by a. In high-copy and low-copy transgenic lines, we found correct temporal and spatial expression of COL6A1 mRNA, paralleling the expression of the murine Col6a1 gene in a panel of nine adult and four fetal organs. It is shown that the DRD1-rtTA BAC-driven expression of a tet-operated reporter is under tight regulation by doxycycline . . If the bacteria do not form colonies inside the patient, the person must repeatedly ingest the modified bacteria in order to get the required doses. Yeast artificial chromosomes is a human engineered DNA molecule used to clone DNA sequences in yeast cells. To meet these requirements large constructs based on bacterial artificial chromosomes (BACs) have been successfully used. Summary Thirty years after the first transgenic mouse was produced, a plethora of genetic tools has been developed to study immune cells in vivo. To explore whether mutant parkin could exert similar pathogenic effects to mammalian DA neurons in vivo, we developed a BAC (bacterial artificial chromosome) transgenic mouse model expressing a C-terminal truncated human mutant parkin (Parkin-Q311X) in DA neurons driven by a dopamine transporter promoter. Defining the Functional Boundaries of the Gata2 Locus by Rescue with a Linked Bacterial Artificial Chromosome Transgene* William . As a model for PD, we generated human α-syn bacterial artificial chromosome transgenic mice (BAC tg mice) harboring the entire human α-syn gene and its gene expression regulatory regions. This paper describes the key elements in the development of the BAC system and its current notable applications. Structure of the modified bacterial artificial chromosome (BAC) containing Prestin and characterization of the transgenic mice.A: Site-directed mutagenesis of a BAC containing the mouse Prestin gene. The strand breaks occur at the approximate positions Bacterial artificial chromosome vectors (BACs) were developed to permit the cloning and stable maintenance of large (100-200 kb) pieces of DNA in E. coli. often alongside transgenic mice. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Edited by: Pradeep Chatterjee. 3. (A) Relative position of the hFOXP3 gene is indicated by thick line, positions of the hFOXP3 promoter, hCNS1, hCNS2, and the last exon (hExon11) are also indicated. Their stability and ease of handling have made these vectors increasingly popular for whole genome mapping and sequencing projects from microbes, plants, and animals. [1][2][3] F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. Enabling the bacteria to form a colony could provide a more long-term solution . In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC) encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. [1] [2] F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division.The bacterial artificial chromosome's usual insert size is 150 . Apolipoprotein B gene expression in a series of human apolipoprotein B transgenic mice generated with recA-assisted restriction endonuclease cleavage-modified bacterial artificial chromosomes: an intestine-specific enhancer element is located between 54 and 62 kilobases 5′ to the structural gene. Defining the detailed expression patterns and developmental regulation of RGS proteins has been hampered by an absence of antibodies useful for mapping. BAC transgenesis is a powerful tool for the study of gene expression and gene function in the mouse in vivo. BACs are often used in connection with DNA sequencing. The Lysogenic Mode The site-specific recombination process that inserts/excises phage A. DNA into/from the chromosome of its E. coli host. Nat. BACs have been . The present work presents a novel in vivo DRD1-Bacterial Artificial Chromosome (BAC) Tet-on system allowing for the inducible activation of tet-operated transgenes specifically within DRD1-expressing cells of transgenic mice. and to provide a library of verified bacterial artificial chromosome (BAC) vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and . Human bacterial artificial chromosome (BAC) transgenesis fully rescues noradrenergic function in dopamine β-hydroxylase knockout mice Joseph F. Cubells, Jason P. Schroeder, Elizabeth S. Barrie, Daniel F. Manvich , Wolfgang Sadee, Tiina Berg, Kristina Mercer, Taylor A. Stowe, L. Cameron Liles, Katherine E. Squires, Andrew Mezher, Patrick Curtin . BACs contain large segments of chromosomal DNA (over 100,000 base pairs). . Enkephalins are endogenous opiates that are assumed to modulate nociceptive information by mediating synaptic transmission in the central nervous system, including the spinal dorsal horn. Exchange occurs between the phage attP site (red) and the bacterial attB site (blue), and the prophage attL and attR sites. Bacterial artificial chromosome A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. Bacterial artificial chromosome (BAC) vectors, which can accommodate large genomic intervals of up to 300 kb are appropriate for such studies (1). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a membrane localized cherry red fluorescent protein (mCherry), driven by the full‐length SMA . BAC-Cre constructs for 10 genes ( Chat , Th , Slc6a4 , Slc6a2 , Etv1 , Ntsr1 , Drd2 , Drd1 , Pcp2 , and Cmtm5 ) produced 14 lines with Cre expression in specific neuronal and glial populations in the brain. These results indicate that the Cd72 polymorphism affects susceptibility to lupus phenotypes and that novel functional rescue by a bacterial artificial chromosome transgenesis is an efficient . Bacterial artificial chromosome (BAC) vectors, which can accommodate large genomic intervals of up to 300 kb are appropriate for such studies (1). The number of splenic CD4-CD8-T cells in Cd72 B6 tg mice was decreased significantly in association with a reduced response to B cell receptor signaling. Physiological Genomics, 2006. The IRES/Cre cassette contains an additional XbaI (X) site. Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. Biotechnol. This unit provides a comprehensive overview on the generation of transgenic mice using bacterial artificial chromosomes (BACs), and the application of BAC transgenic mice in neuroscience research. Figure 2. Bacterial Artificial Chromosomes provides a comprehensive collection of the protocols and resources developed for BACs in recent . J Biol Chem 273 1998 2180021807 The chromosome 21 gene RCAN1, encoding a modulator of the calcineurin (CaN) phosphatase, is a candidate gene for contributing to cognitive disability in people with Down syndrome (DS; trisomy 21). In Bacterial Artificial Chromosomes, expert investigators describe not only the classic methods, but also the many novel techniques they have perfected for the transfer of large DNAs into the cells of both microbes and animals via large-insert recombinant DNAs. Transgenic mice are among the most helpful tools to study the role of genes in physiological conditions. (A) A bacterial artificial chromosome (BAC) containing wild-type human PRSS1 and PRSS2 genes was used to generate PRSS1-PRSS2 transgenic mice (top).For PRSS1 R122H-PRSS2 transgenic mice, an R122H single-point mutation was introduced into the BAC by Galk-mediated . BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons. Although wild-type YACs are often used for this purpose, the inherently high frequency of homologous recombination in yeast makes it possible to precisely modify YACs by deletion or insertion of exogenous DNA. Nkx2-5 BAC-GFP expression was copy number-dependent and locus site-independent. Noncoding expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. After screening a bacterial artificial chromosome of human genomic DNA library with human HS-40, ζ-, α-, and θ-globin probes, a 110-kb clone bearing the whole human α-globin gene cluster was obtained and rare restriction endonuclease mapping was performed. Yang, X., Model, P. & Heintz, N. Homologous recombination based modification in Esherichia coli and germline transmission in transgenic mice of a bacterial artificial chromsome. Comprehensive and cutting-edge, the two volumes of Bacterial Artificial Chromosomes provide a superlative collection of highly productive protocols that will prove useful to many bioscientists,. 3,673. Next, rived artificial chromosomes [PACs], and yeast artificial it was reported that live mouse spermatozoa incubated with chromosomes [YACs]) in transgenesis has become a valid exogenous DNA could result in transgenic offspring [10]. Gene expression from bacterial artificial chromosome (BAC) clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. By John J. Armstrong and Karen K. Hirschi. We have utilized bacterial artificial chromosome (BAC) methods to create transgenic mice that express GFP under the control of endogenous regulator of G-protein signaling 4 (RGS4) enhancer . Here we report transgenic mice carrying a bacterial artificial chromosome (BAC) containing the full human C9orf72 gene with either a normal allele (15 repeats) or disease . 12 Generation of BAC Transgenic Mice, Vikki M. Marshall, Jan Allison, Tanya Templeton and Simon J. Foote; 13 BAC Rescue: A Tool for Functional Analysis of The Mouse Genome, Deborah A. (B,C) DNA isolated from hFOXP3/AmCyan reporter cells (hFOXP3/EL4), hFOXP3/AmCyan transgenic mouse . Transgenic microbes have also been used in recent research to kill or hinder tumors, and to fight Crohn's disease. Expression of human smooth muscle calponin in transgenic mice revealed with a bacterial artificial chromosome Joseph M. Miano , Chad M. Kitchen, Jiyuan Chen, Kathleen M. Maltby, Louise A. Kelly, Hartmut Weiler, Ralf Krahe, Linda K. Ashworth, Emilio Garcia Bacterial artificial chromosome (BAC) transgenic mice harboring the human BDNF gene and its regulatory flanking sequences constitute a useful tool for studying human BDNF gene regulation and for identification of therapeutic compounds modulating BDNF expression. Defining the Deletion Size in Williams-Beuren Syndrome by Fluorescent In Situ . . Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. The bacterial artificial chromosome DNA was isolated, and transgenic mice were generated. Three founders were detected from 35 newborn . Recent technological developments allow the transfer of YACs into mouse embryonal stem (ES) cells and the subsequent generation of transgenic mice. Bacterial artificial chromosome transgenic mice expressing a truncated mutant parkin exhibit age-dependent hypokinetic motor deficits, dopaminergic neuron degeneration, and accumulation of proteinase K-resistant alpha-synuclein. Definition A bacterial artificial chromosome (BAC) is an engineered DNA molecule used to clone DNA sequences in bacterial cells (for example, E. coli). . The bacterial artificial chromosome-dopamine transporter-iCre transgenic line is a unique tool for targeting Cre/loxP-mediated DNA recombination to dopamine neurons for studies of gene function or for labeling living cells, following the crossing of these mice with transgenic Cre reporter lines producing fluorescent proteins. Smooth muscle α actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells (vascular smooth muscle cells and pericytes). Transgenic mouse lines generated from this modified BAC clone closely resembled the endogenous Nkx2-5 expression in the heart, pylorus sphincter, and spleen, but expression was not detected in the tongue. Transgenic mice have been produced with yeast artificial chromosomes (YACs) containing a variety of gene loci (5, 6). 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