SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

Tan, Guoqiang; Gao, Yin; Miao, Shun; Zhang, Xinyue; He, Shuchang; Chen, Zhangliang; Chen, An

doi:10.1093/nar/gni124

Cited by 140 publications

(137 citation statements)

References 27 publications

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“…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

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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...

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Section: Methodsmentioning

confidence: 99%

In planta gene targeting

Fauser

Roth

Pacher

et al. 2012

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

196

176

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“…Approximately 0.1 g of the sample was ground to a fine powder in liquid nitrogen, and the NtCIPK2 promoter was isolated by SiteFinding PCR, as previously described (Tan et al, 2005). PLACE (http://www.dna.affrc.go.jp/PLACE/signalup.html) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) were used to analyze cis-regulatory elements.…”

Section: Isolation Of Ntcipk2 Promotermentioning

confidence: 99%

Molecular cloning and functional characterization of a novel CBL-interacting protein kinase NtCIPK2 in the halophyte Nitraria tangutorum

et al. 2014

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ABSTRACT. CBL-interacting protein kinases (CIPKs) mediate many plant responses to abiotic stress. However, their functions are poorly understood in halophytes. In this study, we isolated a CIPK gene, NtCIPK2, from the halophyte Nitraria tangutorum. By sequence alignment and the construction of a phylogenetic tree, we found that NtCIPK2 is similar to CIPK2 proteins from other plants, and contains conserved domains and motifs. The promoter of NtCIPK2 harbors many cis-acting elements that might be recognized and bound by transcription factors that are related to hormones and stress responses. NtCIPK2 was ubiquitously and robustly expressed in all tested organs, and was induced by salinity, drought, heat, and cold stress. The overexpression of NtCIPK2 in Escherichia coli caused better growth against high salinity, alkalinity, and osmotic conditions, dehydration, and extreme temperatures (i.e., heat and cold) compared to the control. 4717©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 13 (3): 4716-4728 (2014) Cloning and characterization of the NtCIPK2 gene Thus, NtCIPK2 is a candidate gene that might improve the stress tolerance of crops and herbs through genetic manipulation.

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“…The promoter sequence was obtained using the sitefinding-PCR method described by Tan et al (2005). The used primers were listed in Supplemental Table S2.…”

Section: Promoter Sequence Isolationmentioning

confidence: 99%

TaCHP: A Wheat Zinc Finger Protein Gene Down-Regulated by Abscisic Acid and Salinity Stress Plays a Positive Role in Stress Tolerance

Zhao

et al. 2010

Plant Physiology

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The plant response to abiotic stresses involves both abscisic acid (ABA)-dependent and ABA-independent signaling pathways. Here we describe TaCHP, a CHP-rich (for cysteine, histidine, and proline rich) zinc finger protein family gene extracted from bread wheat (Triticum aestivum), is differentially expressed during abiotic stress between the salinity-sensitive cultivar Jinan 177 and its tolerant somatic hybrid introgression cultivar Shanrong No.3. TaCHP expressed in the roots of seedlings at the three-leaf stage, and the transcript localized within the cells of the root tip cortex and meristem. TaCHP transcript abundance was higher in Shanrong No.3 than in Jinan 177, but was reduced by the imposition of salinity or drought stress, as well as by the exogenous supply of ABA. When JN17, a salinity hypersensitive wheat cultivar, was engineered to overexpress TaCHP, its performance in the face of salinity stress was improved, and the ectopic expression of TaCHP in Arabidopsis (Arabidopsis thaliana) also improved the ability of salt tolerance. The expression level of a number of stress reporter genes (AtCBF3, AtDREB2A, AtABI2, and AtABI1) was raised in the transgenic lines in the presence of salinity stress, while that of AtMYB15, AtABA2, and AtAAO3 was reduced in its absence. The presence in the upstream region of the TaCHP open reading frame of the cis-elements ABRE, MYBRS, and MYCRS suggests that it is a component of the ABA-dependent and -independent signaling pathways involved in the plant response to abiotic stress. We suggest that TaCHP enhances stress tolerance via the promotion of CBF3 and DREB2A expression.

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SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

Cited by 140 publications

References 27 publications

In planta gene targeting

In planta gene targeting

Molecular cloning and functional characterization of a novel CBL-interacting protein kinase NtCIPK2 in the halophyte Nitraria tangutorum

TaCHP: A Wheat Zinc Finger Protein Gene Down-Regulated by Abscisic Acid and Salinity Stress Plays a Positive Role in Stress Tolerance

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