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Bubner, Ben; Gase, Klaus; Baldwin, Ian Thomas

doi:10.1186/1472-6750-4-14

Cited by 146 publications

(61 citation statements)

References 16 publications

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“…Although qPCR has previously been used to identify homozygous plants in early generations [7][8][9]18], none of the previously reported qPCR methods can simultaneously achieve high accuracy, reliability, and simplicity for determining transgene homozygosis and transgene stacking (Supplementary Table 2). For example, only 15-46 % of qPCR for copy number determination has been confirmed by Southern blotting analysis in a study involving qPCR screening [18]. In another qPCR-based method, the accuracy only reached 83 % [8].…”

Section: Discussionmentioning

confidence: 98%

Fast-Tracking Determination of Homozygous Transgenic Lines and Transgene Stacking Using a Reliable Quantitative Real-Time PCR Assay

Wang

Jiang

Yang

2014

Appl Biochem Biotechnol

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The selection of homozygous lines is a crucial step in the characterization of newly generated transgenic plants. This is particularly time- and labor-consuming when transgenic stacking is required. Here, we report a fast and accurate method based on quantitative real-time PCR with a rice gene RBE4 as a reference gene for selection of homozygous lines when using multiple transgenic stacking in rice. Use of this method allowed can be used to determine the stacking of up to three transgenes within four generations. Selection accuracy reached 100 % for a single locus and 92.3 % for two loci. This method confers distinct advantages over current transgenic research methodologies, as it is more accurate, rapid, and reliable. Therefore, this protocol could be used to efficiently select homozygous plants and to expedite time- and labor-consuming processes normally required for multiple transgene stacking. This protocol was standardized for determination of multiple gene stacking in molecular breeding via marker-assisted selection.

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Section: Discussionmentioning

confidence: 98%

Fast-Tracking Determination of Homozygous Transgenic Lines and Transgene Stacking Using a Reliable Quantitative Real-Time PCR Assay

Wang

Jiang

Yang

2014

Appl Biochem Biotechnol

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“…Ϫ⌬⌬Ct method (29). The p values for significant differences are provided and indicated by the asterisks.…”

Section: Discussionmentioning

confidence: 99%

RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) β-Exon Splicing

Lee

Cao

Witwicka

et al. 2010

Journal of Biological Chemistry

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Mammalian MBNL (muscleblind-like) proteins are regulators of alternative splicing and have been implicated in myotonic dystrophy, the most common form of adult onset muscular dystrophy. MBNL3 functions as an inhibitor of muscle differentiation and is expressed in proliferating muscle precursor cells but not in differentiated skeletal muscle. Here we demonstrate that MBNL3 regulates the splicing pattern of the muscle transcription factor myocyte enhancer factor 2 (Mef2) by promoting exclusion of the alternatively spliced ␤-exon. Expression of the transcriptionally more active (؉)␤ isoform of Mef2D was sufficient to overcome the inhibitory effects of MBNL3 on muscle differentiation. These data suggest that MBNL3 antagonizes muscle differentiation by disrupting Mef2 ␤-exon splicing. MBNL3 regulates Mef2D splicing by directly binding to intron 7 downstream of the alternatively spliced exon in the pre-mRNA. The RNA binding activity of MBNL3 requires the CX 7 CX 4 -6 CX 3 H zinc finger domains. Using a cell culture model of myotonic dystrophy and myotonic dystrophy patient tissue, we have evidence that expression of CUG expanded RNAs can lead to an increase in MBNL3 expression and a decrease in Mef2D ␤-exon splicing. These studies suggest that elevating MBNL3 activity in myogenic cells could lead to muscle degeneration disorders such as myotonic dystrophy.

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“…Though both approaches were developed for relative copy number estimates, the former is based on a ratio between two targets while the latter quantifies an unknown amount of target towards a standard curve for the same target [22,23]. So far, qPCR has been employed to detect transgene copy number in several plant species, such as Nicotiana tabacum [24], Brassica napus [25], Zea mays hybrids [20], Oryza sativa spp. [26], Gossypium spp.…”

Section: Introductionmentioning

confidence: 99%

Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane

Xue

Guo

Que

et al. 2014

IJMS

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Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

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Cited by 146 publications

References 16 publications

Fast-Tracking Determination of Homozygous Transgenic Lines and Transgene Stacking Using a Reliable Quantitative Real-Time PCR Assay

Fast-Tracking Determination of Homozygous Transgenic Lines and Transgene Stacking Using a Reliable Quantitative Real-Time PCR Assay

RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) β-Exon Splicing

Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane

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