Amplification of genomic sequences flanking transposable elements in host and heterologous plants: a tool for transposon tagging and genome characterization

Earp, David J.; Lowe, Brenda; Baker, Barbara

doi:10.1093/nar/18.11.3271

Cited by 56 publications

(36 citation statements)

References 50 publications

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“…piggyBac insertion sites in transformed parasites were identified by using an inverse PCR technique (29). Genomic DNA (1 g) from drug-resistant parasite populations was digested overnight with 10 units of Sau3AI or RsaI, precipitated with 2 vol of ethanol and 1͞10 vol of 3 M sodium acetate, and self-ligated in a 100-l reaction.…”

Section: Methodsmentioning

confidence: 99%

“…The sites of integration in the transformed populations were identified by inverse PCR analyses performed using inverted oligonucleotide primers within the ITR2 arm of the transposed piggyBac element (29). From the multiple integrations obtained in the transformed populations, we isolated and identified 10 different insertion sites.…”

Section: Insertions Are Stablementioning

confidence: 99%

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High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac

Balu

Shoue

Fraser

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

154

172

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Functional analysis of the Plasmodium falciparum genome is restricted because of the limited ability to genetically manipulate this important human pathogen. We have developed an efficient transposon-mediated insertional mutagenesis method much needed for high-throughput functional genomics of malaria parasites. A drug-selectable marker, human dihydrofolate reductase, added to the lepidopteran transposon piggyBac, transformed parasites by integration into the P. falciparum genome in the presence of a transposase-expressing helper plasmid. Multiple integrations occurred at the expected TTAA target sites throughout the genome of the parasite. We were able to transform P. falciparum with this piggyBac element at high frequencies, in the range of 10 ؊3 , and obtain stable clones of insertional mutants in a few weeks instead of 6 -12 months. Our results show that the piggyBac transposition system can be used as an efficient, random integration tool needed for large-scale, whole-genome mutagenesis of malaria parasites. The availability of such an adaptable genetic tool opens the way for much needed forward genetic approaches to study this lethal human parasite.

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Section: Methodsmentioning

confidence: 99%

Section: Insertions Are Stablementioning

confidence: 99%

High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac

Balu

Shoue

Fraser

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

154

172

View full text Add to dashboard Cite

show abstract

“…PB transposon insertion sites in transformed cells were identified using an inverse PCR technique (22). Genomic DNA (1 μg) from two transformed single clones was isolated using a Genomic DNA purification kit (Promega, Madison, WI, USA) and then digested with HaeIII.…”

Section: Apoptosis and Cell Cycle Analysismentioning

confidence: 99%

High-level transgene expression mediated by the piggyBac transposon enhances transgenic therapeutic effects in cervical cancer xenografts

Kang

Yu²,

Sun³

et al. 2010

Oncol Rep

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Abstract.The piggyBac (PB) transposon is a recently identified, active and flexible transgene vector, combining the advantages of non-viral gene delivery with genomic integration and persistent transgene expression. In this study, we utilized the PB transposon to carry the herpes simplex thymidine kinase (HSV-tk) and red fluorescent protein (mRFP1) reporter genes into the HeLa cervical cancer cell line or tumor xenografts of cervical cancer. Our data showed that HSV-tk and mRFP1 were expressed in HeLa cells and tumor xenografts three weeks after intratumoral injection. The mRNA and protein levels of HSV-tk and mRFP1 were increased by using the PB transposon vector. Our system also demonstrated that sensitivity of transfected HeLa cells to the pro-drug ganciclovir (GCV) was enhanced in vitro and in vivo. Furthermore, our data indicated that the enhanced transgenic therapeutic effect was strongly associated with high-level transgene expression mediated by the PB transposon. Our results suggest that applying the PB transposon in HSV-tk gene delivery and GCV treatment is a promising gene therapy strategy in the treatment of cervical cancer.

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“…By assuming that the Ds sequences were intact, primers corresponding to both ends of Ds were used to amplify sequences flanking Ds::HPT by inverse PCR (IPCR) (Earp et al, 1990;Triglia et al, 1988). Sequences of cloned IPCR products are shown in Figure 5.…”

Section: Transformation Of Rice Protoplasts By Ds::h Ptmentioning

confidence: 99%

Transposition of the maize Ds element from a viral vector to the rice genome

Sugimoto

Otsuki²,

Saji³

et al. 1994

The Plant Journal

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SummaryThe geminivirus miecanthus streak virus (MiSV) was used as a gene vector to study the transposition of the maize D$ element in rice protoplasts. Efficient excision of the Ds from the MiSV vector was observed only when the MiSV vector was allowed to replicate and the plasmid expressing the transposese gene encoded by Ac was co-transfected. Under the same condition, the Ds carrying a hygromycin phosphotransfersse gene (Ds::HPT) was also efficiently excised. Hygromycin-rasistant calli were obtained by culturing these transfected protoplasts in order to examine the transpmdtion of the excimKI Ds::HPTinto the rice genome. In five out of 16 calli examined, the Ds::HPT, but not the vector sequence, was integrated into the rice genome and 8 bp target site duplication typical of AciDs transposition was generated. These results show that the D$::HPT inserted in the MiSV vector transposed directly into the rice genome. This demonstrates the direct transposition of s cloned plant transposable element into the plant genome. Implications of these finding are discussed.

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Amplification of genomic sequences flanking transposable elements in host and heterologous plants: a tool for transposon tagging and genome characterization

Cited by 56 publications

References 50 publications

High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac

High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac

High-level transgene expression mediated by the piggyBac transposon enhances transgenic therapeutic effects in cervical cancer xenografts

Transposition of the maize Ds element from a viral vector to the rice genome

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