An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome

O’Malley, Ronan; Alonso, José M.; Kim, Christopher J.; Leisse, Thomas J.; Ecker, Joseph R.

doi:10.1038/nprot.2007.425

Cited by 112 publications

(101 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The PCR primers amplify sequences in the left ITR of SB; thus, this protocol can be universally applied irrespective of the gene of interest that was cloned in the SB vector. In order to assess copy numbers of integrated transposons and map the genomic integration sites, a ligation-mediated PCR procedure is applied 56 . The procedure consists of a restriction enzyme digest of the genomic DNA, ligation of an oligonucleotide adapter to the ends of the fragmented DNA, PCR amplification of a transgene/genomic DNA junction in two rounds of nested PCR with primers specific to the adapter and to the ITRs of the SB transposon, and sequencing of the junctions to map the insertion to the reference genome 57 .…”

Section: Experimental Designmentioning

confidence: 99%

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

et al. 2014

View full text Add to dashboard Cite

2The pig has emerged as an important large animal model in biomedical and pharmaceutical research.We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in pigs by using the Sleeping Beauty transposon system. The protocol is based on co-injection of a plasmid encoding the SB100X hyperactive transposase together with a second plasmid carrying a transgene flanked by binding sites for the transposase, into the cytoplasm of porcine zygotes. The transposase mediates excision of the transgene cassette from the plasmid vector and its permanent insertion into the genome to produce stable transgenic animals. This method compares favorably in terms of both efficiency and reliable transgene expression to classic pronuclear microinjection or somatic cell nuclear transfer, and offers comparable efficacies to lentiviral approaches, without limitations on vector design, issues of transgene silencing as well as the toxicity and biosafety concerns of working with viral vectors. Microinjection of the vectors into zygotes and transfer of the embryos to recipient animals can be performed in one day; generation of germline-transgenic lines by using this protocol takes approximately one year.

show abstract

Section: Experimental Designmentioning

confidence: 99%

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

et al. 2014

View full text Add to dashboard Cite

show abstract

“…Single-copy plant lines were identified by Southern blot analysis. Insertion junctions were determined using SiteFinder (35), inverse PCR (36, 37), or adapter ligation-mediated PCR protocol (38).…”

Section: Methodsmentioning

confidence: 99%

In planta gene targeting

Fauser

Roth

Pacher

et al. 2012

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

196

176

View full text Add to dashboard Cite

The development of designed site-specific endonucleases boosted the establishment of gene targeting (GT) techniques in a row of different species. However, the methods described in plants require a highly efficient transformation and regeneration procedure and, therefore, can be applied to very few species. Here, we describe a highly efficient GT system that is suitable for all transformable plants regardless of transformation efficiency. Efficient in planta GT was achieved in Arabidopsis thaliana by expression of a sitespecific endonuclease that not only cuts within the target but also the chromosomal transgenic donor, leading to an excised targeting vector. Progeny clonal for the targeted allele could be obtained directly by harvesting seeds. Targeted events could be identified up to approximately once per 100 seeds depending on the target donor combination. Molecular analysis demonstrated that, in almost all events, homologous recombination occurred at both ends of the break. No ectopic integration of the GT vector was found.plant biotechnology | plant breeding | gene technology | double-strand-break repair S ince the first report on gene targeting (GT) in plants was published (1), various approaches were tested to improve the efficiency of the method (2-5), which has been summarized in recent reviews (6, 7). We were able to demonstrate that the integration of a transfer DNA (T-DNA) by homologous recombination (HR) into a specific locus could be enhanced by two orders of magnitude via double-strand-break (DSB) induction using a site-specific endonuclease (8). More recently, by the use of zinc finger nucleases (9), which, in principle, can be used to induce a DSB at any genomic site, endogenous loci have been targeted in Arabidopsis (10), tobacco (11), and maize (12) at high frequencies (13). Some time ago, a method for in vivo targeting was developed in Drosophila. A stably integrated donor precursor molecule is first circularized by the FLP recombinase and subsequently linearized via cutting a single I-SceI recognition site, generating the actual GT vector (14). However, such a technique has not been successfully transferred to plants. We have previously shown that DNA can be efficiently excised from the genome in planta by the use of a site-specific endonuclease (15). To test whether the combination of this approach with DSB-induced recombination might lead to an efficient GT system, that is independent of transformation, we performed a proof-of-concept (POC) experiment in Arabidopsis using I-SceI (16) as a sitespecific nuclease. ResultsGenerating Homozygous Single-Copy GT Lines. Our in planta GT system is based on three different constructs (two shown in Fig. 1A) that were transformed independently by floral dipping. The target locus contains a truncated β-glucuronidase (GUS) gene (uidA) that can be restored via GT. DSB induction at the two ISceI recognition sites flanking a kanamycin-resistance gene would result in excision of the kanamycin-resistance gene and in activation of the target locus for HR. Th...

show abstract

“…Amplification of the pHYG3 insertion junction was performed according to a previously described strategy (O'Malley et al, 2007) using genomic DNA prepared as described (Umen and Goodenough, 2001). Genomic DNA from 266F3 was digested with PvuII, SmaI, StuI, and PmlI and blunt-end ligated to annealed adaptor oligonucleotides (Supplemental Table 1).…”

Section: Vip1-1 Mapping and Genotypingmentioning

confidence: 99%

Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas

et al. 2016

View full text Add to dashboard Cite

The networks that govern carbon metabolism and control intracellular carbon partitioning in photosynthetic cells are poorly understood. Target of Rapamycin (TOR) kinase is a conserved growth regulator that integrates nutrient signals and modulates cell growth in eukaryotes, though the TOR signaling pathway in plants and algae has yet to be completely elucidated. We screened the unicellular green alga Chlamydomonas reinhardtii using insertional mutagenesis to find mutants that conferred hypersensitivity to the TOR inhibitor rapamycin. We characterized one mutant, vip1-1, that is predicted to encode a conserved inositol hexakisphosphate kinase from the VIP family that pyrophosphorylates phytic acid (InsP 6 ) to produce the low abundance signaling molecules InsP 7 and InsP 8 . Unexpectedly, the rapamycin hypersensitive growth arrest of vip1-1 cells was dependent on the presence of external acetate, which normally has a growth-stimulatory effect on Chlamydomonas. vip1-1 mutants also constitutively overaccumulated triacylglycerols (TAGs) in a manner that was synergistic with other TAG inducing stimuli such as starvation. vip1-1 cells had reduced InsP 7 and InsP 8 , both of which are dynamically modulated in wild-type cells by TOR kinase activity and the presence of acetate. Our data uncover an interaction between the TOR kinase and inositol polyphosphate signaling systems that we propose governs carbon metabolism and intracellular pathways that lead to storage lipid accumulation.

show abstract

An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome

Cited by 112 publications

References 7 publications

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

In planta gene targeting

Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas

Contact Info

Product

Resources

About