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https://www.readbyqxmd.com/read/29617873/a-multi-landing-pad-dna-integration-platform-for-mammalian-cell-engineering
#1
Leonid Gaidukov, Liliana Wroblewska, Brian Teague, Tom Nelson, Xin Zhang, Yan Liu, Kalpana Jagtap, Selamawit Mamo, Wen Allen Tseng, Alexis Lowe, Jishnu Das, Kalpanie Bandara, Swetha Baijuraj, Nevin M Summers, Timothy K Lu, Lin Zhang, Ron Weiss
Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three 'landing pad' recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection...
May 4, 2018: Nucleic Acids Research
https://www.readbyqxmd.com/read/29188179/development-of-a-high-efficiency-integration-system-and-promoter-library-for-rapid-modification-of-pseudomonas-putida-kt2440
#2
Joshua R Elmore, Anna Furches, Gara N Wolff, Kent Gorday, Adam M Guss
Pseudomonas putida strains are highly robust bacteria known for their ability to efficiently utilize a variety of carbon sources, including aliphatic and aromatic hydrocarbons. Recently, P. putida has been engineered to valorize the lignin stream of a lignocellulosic biomass pretreatment process. Nonetheless, when compared to platform organisms such as Escherichia coli , the toolkit for engineering P. putida is underdeveloped. Heterologous gene expression in particular is problematic. Plasmid instability and copy number variance provide challenges for replicative plasmids, while use of homologous recombination for insertion of DNA into the chromosome is slow and laborious...
December 2017: Metabolic Engineering Communications
https://www.readbyqxmd.com/read/28815508/multipart-dna-assembly-using-site-specific-recombinases-from-the-large-serine-integrase-family
#3
Femi J Olorunniji, Christine Merrick, Susan J Rosser, Margaret C M Smith, W Marshall Stark, Sean D Colloms
Assembling multiple DNA fragments into functional plasmids is an important and often rate-limiting step in engineering new functions in living systems. Bacteriophage integrases are enzymes that carry out efficient recombination reactions between short, defined DNA sequences known as att sites. These DNA splicing reactions can be used to assemble large numbers of DNA fragments into a functional circular plasmid in a method termed serine integrase recombinational assembly (SIRA). The resulting DNA assemblies can easily be modified by further recombination reactions catalyzed by the same integrase in the presence of its recombination directionality factor (RDF)...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28335006/a-platform-for-functional-assessment-of-large-variant-libraries-in-mammalian-cells
#4
Kenneth A Matreyek, Jason J Stephany, Douglas M Fowler
Sequencing-based, massively parallel genetic assays have revolutionized our ability to quantify the relationship between many genotypes and a phenotype of interest. Unfortunately, variant library expression platforms in mammalian cells are far from ideal, hindering the study of human gene variants in their physiologically relevant cellular contexts. Here, we describe a platform for phenotyping variant libraries in transfectable mammalian cell lines in two steps. First, a landing pad cell line with a genomically integrated, Tet-inducible cassette containing a Bxb1 recombination site is created...
June 20, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28317293/a-new-location-to-split-cre-recombinase-for-protein-fragment-complementation
#5
Maryam Rajaee, David W Ow
We have previously described a recombinase-mediated gene stacking system in which the Cre recombinase is used to remove lox-site flanked DNA no longer needed after each round of Bxb1 integrase-mediated site-specific integration. The Cre recombinase can be conveniently introduced by hybridization with a cre-expressing plant. However, maintaining an efficient cre-expressing line over many generations can be a problem, as high production of this DNA-binding protein might interfere with normal chromosome activities...
November 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28286726/multiple-expression-cassette-exchange-via-tp901-1-r4-and-bxb1-integrase-systems-on-a-mouse-artificial-chromosome
#6
Kosuke Tomimatsu, Kenji Kokura, Tadashi Nishida, Yuki Yoshimura, Yasuhiro Kazuki, Masashi Narita, Mitsuo Oshimura, Tetsuya Ohbayashi
The site-specific excision of a target DNA sequence for genetic knockout or lineage tracing is a powerful tool for investigating biological systems. Currently, site-specific recombinases (SSRs), such as Cre or Flp recombination target cassettes, have been successfully excised or inverted by a single SSR to regulate transgene expression. However, the use of a single SSR might restrict the complex control of gene expression. This study investigated the potential for expanding the multiple regulation of transgenes using three different integrase systems (TP901-1, R4, and Bxb1)...
March 2017: FEBS Open Bio
https://www.readbyqxmd.com/read/28186334/a-novel-bxb1-integrase-rmce-system-for-high-fidelity-site-specific-integration-of-mab-expression-cassette-in-cho-cells
#7
Mara C Inniss, Kalpanie Bandara, Barbara Jusiak, Timothy K Lu, Ron Weiss, Liliana Wroblewska, Lin Zhang
As CHO cell line development for biotherapeutic production becomes more sophisticated through the availability of the CHO genome sequence, the ability to accurately and reproducibly engineer the host cell genome has become increasingly important. Multiple well characterized systems for site-specific integration will enable more complex cell line engineering to generate cell lines with desirable attributes. We built and characterized a novel recombinase mediated cassette exchange (RMCE) system using Bxb1 integrase and compared it to the commonly used Flp/FRT RMCE system...
August 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28118814/bxb1-phage-recombinase-assists-genome-engineering-in-drosophila-melanogaster
#8
Roumen Voutev, Richard S Mann
Rapid and reliable genome modifications provide the basis for detailed in vivo functional analysis of any genomic entity (gene, regulatory DNA, non-coding RNA, etc). With the advent of CRISPR/Cas9 genome editing technology, manipulation of a particular genomic locus has become a routine undertaking in variety of model organisms, including the fruit fly Drosophila melanogaster . To further diversify the available tools for genome engineering, we successfully harnessed the phage recombinase Bxb1 to perform recombinase-mediated cassette exchange (RMCE) in D...
January 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/27864282/bxb1-integrase-serves-as-a-highly-efficient-dna-recombinase-in-rapid-metabolite-pathway-assembly
#9
Xianwei Wang, Biao Tang, Yu Ye, Yayi Mao, Xiaolai Lei, Guoping Zhao, Xiaoming Ding
Phage-encoded serine integrases are widely used in genetic engineering. They also have the potential to serve as efficient DNA assemblers, demonstrated by the method of site-specific recombination-based tandem assembly (SSRTA) that can combine biological parts into devices, pathways, and systems. Here, four serine integrases, ϕBT1, TG1, ϕRv1, and Bxb1, were investigated to ascertain their in vitro DNA assembly activities. Bxb1 integrase displayed the highest efficiency to obtain final products. Thus, we conclude that Bxb1 integrase is an excellent choice for DNA assembly in vitro Using this enzyme and its recognition sites, BioBrick standards were designed that are compatible with the SSRTA method for module addition...
January 2017: Acta Biochimica et Biophysica Sinica
https://www.readbyqxmd.com/read/27557683/method-for-biolistic-site-specific-integration-in-plants-catalyzed-by-bxb1-integrase
#10
Ruyu Li, Zhiguo Han, Lili Hou, Gurminder Kaur, Qian Yin, David W Ow
Crop improvement is a never ending process. With a transgenesis approach, it is not inconceivable to envision a continuous addition of new transgenes to existing cultivars. Previously, we described a recombinase-directed gene stacking method in tobacco (Hou et al., Mol Plant 7:1756-1765, 2014). Being able to stack DNA to a previous location ensures that the number of genetic loci does not increase with each new round of transgene addition. Whereas the previous demonstration was conducted through polyethylene glycol to mediate uptake of DNA into tobacco protoplasts, we now describe protocols for using biolistic transformation to stack DNA in tobacco and rice...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27550179/crossover-site-sequence-and-dna-torsional-stress-control-strand-interchanges-by-the-bxb1-site-specific-serine-recombinase
#11
Ross A Keenholtz, Nigel D F Grindley, Graham F Hatfull, John F Marko
DNA segment exchange by site-specific serine recombinases (SRs) is thought to proceed by rigid-body rotation of the two halves of the synaptic complex, following the cleavages that create the two pairs of exchangeable ends. It remains unresolved how the amount of rotation occurring between cleavage and religation is controlled. We report single-DNA experiments for Bxb1 integrase, a model SR, where dynamics of individual synapses were observed, using relaxation of supercoiling to report on cleavage and rotation events...
October 14, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/26940434/inheritance-of-steroid-independent-male-sexual-behavior-in-male-offspring-of-b6d2f1-mice
#12
Christine M McInnis, Paul J Bonthuis, Emilie F Rissman, Jin Ho Park
The importance of gonadal steroids in modulating male sexual behavior is well established. Individual differences in male sexual behavior, independent of gonadal steroids, are prevalent across a wide range of species, including man. However, the genetic mechanisms underlying steroid-independent male sexual behavior are poorly understood. A high proportion of B6D2F1 hybrid male mice demonstrates steroid-independent male sexual behavior (identified as "maters"), providing a mouse model that opens up avenues of investigation into the mechanisms regulating male sexual behavior in the absence of gonadal hormones...
April 2016: Hormones and Behavior
https://www.readbyqxmd.com/read/26860416/comparison-and-optimization-of-ten-phage-encoded-serine-integrases-for-genome-engineering-in-saccharomyces-cerevisiae
#13
Zhengyao Xu, William R A Brown
BACKGROUND: Phage-encoded serine integrases, such as ϕC31 integrase, are widely used for genome engineering but have not been optimized for use in Saccharomyces cerevisiae although this organism is a widely used organism in biotechnology. RESULTS: The activities of derivatives of fourteen serine integrases that either possess or lack a nuclear localization signal were compared using a standardized recombinase mediated cassette exchange reaction. The relative activities of these integrases in S...
February 9, 2016: BMC Biotechnology
https://www.readbyqxmd.com/read/26007658/a-modular-open-platform-for-systematic-functional-studies-under-physiological-conditions
#14
Christopher B Mulholland, Martha Smets, Elisabeth Schmidtmann, Susanne Leidescher, Yolanda Markaki, Mario Hofweber, Weihua Qin, Massimiliano Manzo, Elisabeth Kremmer, Katharina Thanisch, Christina Bauer, Pascaline Rombaut, Franz Herzog, Heinrich Leonhardt, Sebastian Bultmann
Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3 and Uhrf1, we generated MIN-tagged embryonic stem cell lines and created a toolbox of prefabricated modules that can be integrated via Bxb1-mediated recombination...
September 30, 2015: Nucleic Acids Research
https://www.readbyqxmd.com/read/25408400/using-phage-integrases-in-a-site-specific-dual-integrase-cassette-exchange-strategy
#15
Jonathan M Geisinger, Michele P Calos
ΦC31 integrase, a site-specific large serine recombinase, is a useful tool for genome engineering in a variety of eukaryotic species and cell types. ΦC31 integrase performs efficient recombination between its attB site and either its own placed attP site or a partially mismatched genomic pseudo attP site. Bxb1 integrase, another large serine recombinase, has a similar level of recombinational activity, but recognizes only its own attB and attP sites. Previously, we have used these integrases sequentially to integrate plasmid DNA into the genome...
2015: Methods in Molecular Biology
https://www.readbyqxmd.com/read/25303219/a-novel-system-for-simultaneous-or-sequential-integration-of-multiple-gene-loading-vectors-into-a-defined-site-of-a-human-artificial-chromosome
#16
Teruhiko Suzuki, Yasuhiro Kazuki, Mitsuo Oshimura, Takahiko Hara
Human artificial chromosomes (HACs) are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV) into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system) via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase...
2014: PloS One
https://www.readbyqxmd.com/read/25281665/an-open-source-system-for-in-planta-gene-stacking-by-bxb1-and-cre-recombinases
#17
Lili Hou, Yuan-Yeu Yau, Junjie Wei, Zhiguo Han, Zhicheng Dong, David W Ow
The rapid development of crops with multiple transgenic traits arouses the need for an efficient system for creating stacked cultivars. Most major crops rely on classical breeding to introgress the transgene from a laboratory variety to the numerous cultivars adapted to different growing regions. Even with vegetative propagated crops, genetic crosses are conducted during varietal improvement prior to vegetative cloning. The probability to assort the 'x' number of transgenic loci into a single genome may seem trivial, (¼) (x) for a diploid species, but given the 'y' number of other nontransgenic traits that breeders also need to assemble into the same genome, the (¼) (x+y) probability for a 'breeding stack' could quickly make the line conversion process unmanageable...
December 2014: Molecular Plant
https://www.readbyqxmd.com/read/25148894/transgene-autoexcision-in-switchgrass-pollen-mediated-by-the-bxb1-recombinase
#18
Maria N Somleva, Chang Ai Xu, Kieran P Ryan, Roger Thilmony, Oliver Peoples, Kristi D Snell, James Thomson
BACKGROUND: Switchgrass (Panicum virgatum L.) has a great potential as a platform for the production of biobased plastics, chemicals and energy mainly because of its high biomass yield on marginal land and low agricultural inputs. During the last decade, there has been increased interest in the genetic improvement of this crop through transgenic approaches. Since switchgrass, like most perennial grasses, is exclusively cross pollinating and poorly domesticated, preventing the dispersal of transgenic pollen into the environment is a critical requisite for the commercial deployment of this important biomass crop...
2014: BMC Biotechnology
https://www.readbyqxmd.com/read/24781921/recombinase-mediated-reprogramming-and-dystrophin-gene-addition-in-mdx-mouse-induced-pluripotent-stem-cells
#19
Chunli Zhao, Alfonso P Farruggio, Christopher R R Bjornson, Christopher L Chavez, Jonathan M Geisinger, Tawny L Neal, Marisa Karow, Michele P Calos
A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC) may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome...
2014: PloS One
https://www.readbyqxmd.com/read/24304893/dice-an-efficient-system-for-iterative-genomic-editing-in-human-pluripotent-stem-cells
#20
Fangfang Zhu, Matthew Gamboa, Alfonso P Farruggio, Simon Hippenmeyer, Bosiljka Tasic, Birgitt Schüle, Yanru Chen-Tsai, Michele P Calos
To reveal the full potential of human pluripotent stem cells, new methods for rapid, site-specific genomic engineering are needed. Here, we describe a system for precise genetic modification of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We identified a novel human locus, H11, located in a safe, intergenic, transcriptionally active region of chromosome 22, as the recipient site, to provide robust, ubiquitous expression of inserted genes. Recipient cell lines were established by site-specific placement of a 'landing pad' cassette carrying attP sites for phiC31 and Bxb1 integrases at the H11 locus by spontaneous or TALEN-assisted homologous recombination...
March 2014: Nucleic Acids Research
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