ppv402  (Addgene inc)

Journal: PLoS Pathogens

Article Title: Transposon-mediated Chromosomal Integration of Transgenes in the Parasitic Nematode Strongyloides ratti and Establishment of Stable Transgenic Lines

doi: 10.1371/journal.ppat.1002871

Figure Lengend Snippet: A ) Donor vector pPV254 incorporating a fluorescent reporter gene in which expression of GFP is driven by the Ss-act-2 promoter and terminated by the Ss era-1 3′ UTR. The reporter transgene in pPV254 is flanked by the inverted terminal repeats (ITR) plus internal sequences common to piggyBac transposable elements. B ) Donor vector pPV356, which is like pPV254 in all respects except that the coding sequence is flanked by the gypsy retroviral insulator sequences from Drosophila . C ) Helper vector pPV402 in which expression of the piggyBac transposase gene is driven by Ss-rps-21 promoter and terminated by the Ss-era-1 3′ UTR. D ) Plasmid pPV257 for in vitro transcription of mRNA encoding the piggyBac transposase under the T7 promoter. In lieu of helper vector pPV402, this mRNA was capped, tailed and co-injected with donor vector pPV356 in Experiment 2.

Article Snippet: Finally, the expression cassette, Ss-act-2 p:: gfp :: Ss-era-1 3′UTR from pAJ08 was cloned into the MCS by digestion with Xba I and Hind III and ligation with T4 DNA Ligase to yield pPV356. pPV402 ( ; GenBank accession number, JX013634), the piggyBac transposase helper vector, was made by excising the piggyBac transposase coding sequence from pPV257 (see below) with the restriction enzymes AgeI and AvrII and cloning them into the pAJ50 vector (Addgene Plasmid #14918) from which the gfp coding region had been removed with the same restriction enzymes.

Techniques: Plasmid Preparation, Expressing, Sequencing, Retroviral, In Vitro, Injection

Journal: PLoS Pathogens

Article Title: Transposon-mediated Chromosomal Integration of Transgenes in the Parasitic Nematode Strongyloides ratti and Establishment of Stable Transgenic Lines

doi: 10.1371/journal.ppat.1002871

Figure Lengend Snippet: Heritable transgene expression and establishment of stable transgene-expressing lines in S. ratti as a function of various pairings of donor and helper plasmids incorporating elements of the piggyBac transposon.

Article Snippet: Finally, the expression cassette, Ss-act-2 p:: gfp :: Ss-era-1 3′UTR from pAJ08 was cloned into the MCS by digestion with Xba I and Hind III and ligation with T4 DNA Ligase to yield pPV356. pPV402 ( ; GenBank accession number, JX013634), the piggyBac transposase helper vector, was made by excising the piggyBac transposase coding sequence from pPV257 (see below) with the restriction enzymes AgeI and AvrII and cloning them into the pAJ50 vector (Addgene Plasmid #14918) from which the gfp coding region had been removed with the same restriction enzymes.

Techniques: Expressing

Journal: PLoS Pathogens

Article Title: Transposon-mediated Chromosomal Integration of Transgenes in the Parasitic Nematode Strongyloides ratti and Establishment of Stable Transgenic Lines

doi: 10.1371/journal.ppat.1002871

Figure Lengend Snippet: Southern hybridization of genomic DNA (gDNA) of S. ratti from stably transformed lines probed for the gfp coding sequence. A ) Transgene diagram showing position of the probe used for Southern hybridization analysis and the single restriction site for BsrGI, the enzyme used for restriction digestion of gDNA. B ) Southern blot of BsrGI digests of gDNA from pooled free-living adults from three independent integrated lines (PV2, PV3 and PV4). The positive control lane is a blot of a BsrGI digest of donor plasmid pPV356, and the negative control is a blot of a BsrGI digest of gDNA from non-transformed S. ratti free-living adults ( S.r. gDNA). Note gfp hybridization signals in multiple restriction fragments in gDNA from each of the three integrated lines. A single hybridizing band of 8.1 kb appears as predicted in the positive control digest of pPV356. No gfp -specific signal is detected in the negative control digest of gDNA from non-transformed S. ratti . C ) Gel analysis of PCR products from genomic DNA templates from non-transformed control parasites, parasites from each of three stable lines, PV2, PV3 and PV4, with integrated, stably expressed transgenes and F1 progeny of free-living female worms microinjected with donor plasmid pPV356 and helper plasmid pPV402. Also included are PCR products from control reactions with plasmids pPV356 and pPV254 as templates and from a reaction from which template was omitted. Upper gel image depicts 624 bp amplification products resulting from a forward primer hybridizing to the M13 reverse priming site in the vector and a reverse primer hybridizing within the transposon sequence. Lower gel image is a loading control showing the expected 554 bp amplification product from reactions with primers specific for the constitutively expressed cellular actin-encoding gene Sr-act-2 .

Article Snippet: Finally, the expression cassette, Ss-act-2 p:: gfp :: Ss-era-1 3′UTR from pAJ08 was cloned into the MCS by digestion with Xba I and Hind III and ligation with T4 DNA Ligase to yield pPV356. pPV402 ( ; GenBank accession number, JX013634), the piggyBac transposase helper vector, was made by excising the piggyBac transposase coding sequence from pPV257 (see below) with the restriction enzymes AgeI and AvrII and cloning them into the pAJ50 vector (Addgene Plasmid #14918) from which the gfp coding region had been removed with the same restriction enzymes.

Techniques: Hybridization, Stable Transfection, Transformation Assay, Sequencing, Southern Blot, Positive Control, Plasmid Preparation, Negative Control, Control, Amplification