We are analyzing https://link.springer.com/article/10.1186/1755-8417-1-3.

Title:
High-efficiency Rosa26 knock-in vector construction for Cre-regulated overexpression and RNAi | PathoGenetics
Description:
Introduction Rosa26 is a genomic mouse locus commonly used to knock-in cDNA constructs for ubiquitous or conditional gene expression in transgenic mice. However, the vectors generally used to generate Rosa26 knock-in constructs show instability problems, which have a severe impact on the efficiency of the system. Results We have optimized the cloning procedure to generate targeting vectors for Cre-regulated expression of constructs within several days with minimal hands-on time, thereby enabling high-throughput approaches. We demonstrate that transient expression of Cre still results in expression of the construct, as shown by the expression level and via functional assays. In addition to its well-established possibilities in expressing cDNA constructs, we show that the Rosa26 locus can be used to drive expression of functional miRNA constructs from its endogenous promoter. Conclusion We provide a new high-efficiency cloning system for Rosa26 knock-in constructs to express either cDNA or miRNA fragments. Our system will enable high-throughput approaches for controlled expression of cDNA or miRNA constructs, with the latter providing a potential high-speed alternative for conditional knock-out models.
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Keywords {🔍}

expression, cells, βcatenin, rosa, constructs, vector, cdna, prosadest, article, construct, locus, targeting, mirna, gene, pubmed, figure, human, system, knockin, endogenous, mouse, cre, google, scholar, mice, cas, vectors, clones, file, shown, oct, rnai, generate, cloning, generation, creregulated, additional, analysis, days, authors, data, models, found, targeted, cassette, reaction, lacz, sequence, site, access,

Topics {✒️}

splice acceptor-lox-stop-lox-gateway entry-poly mir-26a-knockout hela cells caggs-creert2-ires-puro construct open access article designated pentr-s33 β-catenin gateway-compatible mir-155-based vector uk/research/hastie/home feeder-free gelatinized plates lox-stop-lox cassette strong negative counter-selection gateway-compatible entry vector ccdb counter-selection gene e14-derived cell line real-time pcr fragments performed real-time pcr quantitative rt-pcr showed combined bp/lr reaction original gene-trap line combined amp/cam resistance noncanonical wnt/fz signaling mirna-expressing prosa26-dest construct full size image β-catenin proto-oncogene pentr-s33 β-catenin efficient genetic engineering fully es-derived embryos enable high-throughput approaches time-consuming breeding schemes multi-nucleated cells suggestive s33y β-catenin allele measure β-catenin activity high-efficiency cloning system gateway-compatible mirna vector article download pdf rosa26-dest-based mirna constructs transgenic rna interference dissect gene-specific phenotypes rna interference based enabling high-throughput approaches 2-gw/emgfp-mir lacz 2-gw/emgfp-mir oct4 bcat-attb/f-myc quantified β-catenin expression desired tissue-specific pattern mutant β-catenin cdna s33y human β-catenin human β-catenin construct undifferentiated embryonic stem embryonic stem cells related subjects

Schema {🗺️}

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         description:Rosa26 is a genomic mouse locus commonly used to knock-in cDNA constructs for ubiquitous or conditional gene expression in transgenic mice. However, the vectors generally used to generate Rosa26 knock-in constructs show instability problems, which have a severe impact on the efficiency of the system. We have optimized the cloning procedure to generate targeting vectors for Cre-regulated expression of constructs within several days with minimal hands-on time, thereby enabling high-throughput approaches. We demonstrate that transient expression of Cre still results in expression of the construct, as shown by the expression level and via functional assays. In addition to its well-established possibilities in expressing cDNA constructs, we show that the Rosa26 locus can be used to drive expression of functional miRNA constructs from its endogenous promoter. We provide a new high-efficiency cloning system for Rosa26 knock-in constructs to express either cDNA or miRNA fragments. Our system will enable high-throughput approaches for controlled expression of cDNA or miRNA constructs, with the latter providing a potential high-speed alternative for conditional knock-out models.
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      headline:High-efficiency Rosa26 knock-in vector construction for Cre-regulated overexpression and RNAi
      description:Rosa26 is a genomic mouse locus commonly used to knock-in cDNA constructs for ubiquitous or conditional gene expression in transgenic mice. However, the vectors generally used to generate Rosa26 knock-in constructs show instability problems, which have a severe impact on the efficiency of the system. We have optimized the cloning procedure to generate targeting vectors for Cre-regulated expression of constructs within several days with minimal hands-on time, thereby enabling high-throughput approaches. We demonstrate that transient expression of Cre still results in expression of the construct, as shown by the expression level and via functional assays. In addition to its well-established possibilities in expressing cDNA constructs, we show that the Rosa26 locus can be used to drive expression of functional miRNA constructs from its endogenous promoter. We provide a new high-efficiency cloning system for Rosa26 knock-in constructs to express either cDNA or miRNA fragments. Our system will enable high-throughput approaches for controlled expression of cDNA or miRNA constructs, with the latter providing a potential high-speed alternative for conditional knock-out models.
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         cDNA Construct
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         Gateway Cloning System
         miRNA Construct
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         Cell Biology
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