Tn&lt;I&gt;5&lt;/I&gt; as a Molecular Genetics Tool: InVitro Transposition and the Coupling of In Vitro Technologies With In Vivo Transposition

Reznikoff, William S.; Goryshin, Igor Y.; Jendrisak, Jerry

doi:10.1385/1-59259-755-6:083

Cited by 22 publications

(21 citation statements)

References 11 publications

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“…1C, step 1). We first used this randomly inserting transposon (Reznikoff et al 2004) to generate a saturated bank of transposon-containing pBSltrB plasmids (pBSltrBTTn) (Fig. 3B, step 1).…”

Section: Resultsmentioning

confidence: 99%

“…The statistical representation of the bank was calculated by dividing the number of Spc colonies recuperated following transformation of the transposition assay (Tn5-containing plasmids) by twice the size of the plasmid. This method of calculation takes into consideration that Tn5 can insert randomly on either strand of the target plasmid (Goryshin and Reznikoff 1998;Reznikoff et al 2004). The pBSltrBTTn bank was used to construct the various pLE12TTn banks except the V-VI bank.…”

Section: Plasmidsmentioning

confidence: 99%

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Trans-splicing versatility of the Ll.LtrB group II intron

Belhocine¹,

Mak²,

Cousineau³

2008

RNA

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Group II introns are found in organelles, bacteria, and archaea. Some harbor an open reading frame (ORF) with reverse transcriptase, maturase, and occasionally endonuclease activities. Group II introns require the assistance of either intronencoded or free-standing maturases to excise from primary RNA transcripts in vivo. Some ORF-containing group II introns were shown to be mobile retroelements that invade new DNA sites by retrohoming or retrotransposition. Group II introns are also hypothesized to be the ancestors of the spliceosome-dependent nuclear introns and the small nuclear RNAs (snRNAs-U1, U2, U4, U5, and U6) that are part of the spliceosome. The ability of some fragmented group II introns to undergo splicing in trans supports the theory that the snRNAs evolved from portions of group II introns. Here, we developed a Tn5-based genetic screen to explore the trans-splicing potential of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis. Proficient trans-splicing variants of Ll.LtrB were selected using a highly sensitive trans-splicing/conjugation screen. We report that numerous fragmentation sites located throughout Ll.LtrB support splicing in trans, showing that this intron is remarkably more tolerant to fragmentation than expected from the fragmentation sites uncovered within natural trans-splicing group II introns. This work unveils the great versatility of group II intron fragments to assemble and accurately trans-splice their flanking exons in vivo.

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“…1C, step 1). We first used this randomly inserting transposon (Reznikoff et al 2004) to generate a saturated bank of transposon-containing pBSltrB plasmids (pBSltrBTTn) (Fig. 3B, step 1).…”

Section: Resultsmentioning

confidence: 99%

Section: Plasmidsmentioning

confidence: 99%

Trans-splicing versatility of the Ll.LtrB group II intron

Belhocine¹,

Mak²,

Cousineau³

2008

RNA

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show abstract

“…Tn5 is a particularly tractable system where the efficiency of the reaction in vitro approaches 100%. The high efficiency of transpososome assembly has facilitated an electroporation technique for generating libraries of transposon insertions (Reznikoff et al 2004). The transpososomes are assembled in vitro where the absence of the catalytic metal ion, Mg 2?…”

Section: Transpososome Preassemblymentioning

confidence: 99%

Gene therapy vectors: the prospects and potentials of the cut-and-paste transposons

Bouuaert

Chalmers

2009

Genetica

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Gene therapy applications require efficient tools for the stable delivery of genetic information into eukaryotic genomes. Most current gene delivery strategies are based on viral vectors. However, a number of drawbacks, such as the limited cargo capacity, host immune response and mutational risks, highlight the need for alternative gene delivery tools. A comprehensive gene therapy tool kit should contain a range of vectors and techniques that can be adapted to different targets and purposes. Transposons provide a potentially powerful approach. However, transposons encompass a large number of different molecular mechanisms, some of which are better suited to gene delivery applications than others. Here, we consider the range and potentials of the various mechanisms, focusing on the cut-and-paste transposons as one of the more promising avenues towards gene therapy applications. Several cut-and-paste transposition systems are currently under development. We will first consider the mechanisms of piggyBac and the hAT family elements Tol1 and Tol2, before focusing on the mariner family elements including Mos1, Himar1 and Hsmar1.

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“…This technique is based on the Tn5 transposition system and produces random insertions in genomic DNA by electroporation of a stable complex between the transposase enzyme and a transposon containing the dihydrofolate reductase (DHFR, resulting in trimethoprim resistance) gene flanked by hyperactive 19 bp mosaic end recognition sequences (Goryshin and Reznikoff 1998;Reznikoff et al 2004). The electroporated cells were immediately recovered in SOC medium for 2 h at 37°C and the colonies were selected on Luria-Bertani (LB) agar containing 10 lg/ml trimethoprim, and frozen at -20°C.…”

Section: Transposon Mutantsmentioning

confidence: 99%

Mutations influencing expression of the Salmonella enterica serovar Enteritidis pathogenicity island I key regulator hilA

Immerseel

Eeckhaut

Boyen

et al. 2008

Antonie van Leeuwenhoek

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Invasion in gut epithelial cells, mediated by genes of the Salmonella pathogenicity island I, is a crucial step in the pathogenesis of Salmonella enterica serovar Enteritidis infections. The most important regulator of the invasive process is the hilA gene. In this study, a transposon bank approach was used to identify DNA sequences affecting expression of hilA. Mutants with decreased hilA expression carried mutations in known virulence gene regulators (fliZ, hilD, sirA), genes encoding ion transport proteins (feoA, feoB, pstB, pstC), genes involved in transcription/translation machinery (nusA, selA) and the hypothetical inner membrane protein STM2303. Mutants yielding increased hilA expression carried a transposon insertion in the known virulence regulator hha, the transcriptional regulator and oxygen sensor fnr and the virulence gene virK. Mutants having decreased and increased hilA expression were more and less invasive in the human colon carcinoma cell line T84 compared to wild type strain bacteria, respectively.

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Tn<I>5</I> as a Molecular Genetics Tool: InVitro Transposition and the Coupling of In Vitro Technologies With In Vivo Transposition

Cited by 22 publications

References 11 publications

Trans-splicing versatility of the Ll.LtrB group II intron

Trans-splicing versatility of the Ll.LtrB group II intron

Gene therapy vectors: the prospects and potentials of the cut-and-paste transposons

Mutations influencing expression of the Salmonella enterica serovar Enteritidis pathogenicity island I key regulator hilA

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