Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants

Biricolti, Stefano; Bogani, Patrizia; Cerboneschi, Matteo; Gori, Massimo

doi:10.1007/s10528-016-9719-z

Cited by 25 publications

(22 citation statements)

References 29 publications

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“…PCR amplification is time and cost consuming in plant preparation and identification. Southern blot analysis, as a traditional method for identification of the transgenic copy number, is costly in terms of reagents, equipment, time and labour [40]. The GUS expression assay is a compromise in the matter of cost.…”

Section: Discussionmentioning

confidence: 99%

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A convenient, rapid and efficient method for establishing transgenic lines of Brassica napus

Zhang

Liu

et al. 2020

Plant Methods

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Background: Brassica napus is an important oilseed crop that offers a considerable amount of biomass for global vegetable oil production. The establishment of an efficient genetic transformation system with a convenient transgenic-positive screening method is of great importance for gene functional analysis and molecular breeding. However, to our knowledge, there are few of the aforementioned systems available for efficient application in B. napus. Results: Based on the well-established genetic transformation system in B. napus, five vectors carrying the red fluorescence protein encoding gene from Discosoma sp. (DsRed) were constructed and integrated into rapeseed via Agrobacterium-mediated hypocotyl transformation. An average of 59.1% tissues were marked with red fluorescence by the visual screening method in tissue culture medium, 96.1% of which, on average, were amplified with the objective genes from eight different rapeseed varieties. In addition, the final transgenic-positive efficiency of the rooted plantlets reached up to 90.7% from red fluorescence marked tissues, which was much higher than that in previous reports. Additionally, visual screening could be applicable to seedlings via integration of DsRed, including seed coats, roots, hypocotyls and cotyledons during seed germination. These results indicate that the highly efficient genetic transformation system combined with the transgenic-positive visual screening method helps to conveniently and efficiently obtain transgenic-positive rapeseed plantlets. Conclusion: A rapid, convenient and highly efficient method was developed to obtain transgenic plants, which can help to obtain the largest proportion of transgene-positive regenerated plantlets, thereby avoiding a long period of plant regeneration. The results of this study will benefit gene functional studies especially in high-throughput molecular biology research.

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Section: Discussionmentioning

confidence: 99%

“…Southern blotting analysis was performed in transgenic rapeseed plants according to the published methods with a minor modification [40]. Briefly, total genomic DNA was extracted using the standard CTAB method to obtain a large amount of DNA.…”

Section: Dna Extraction Pcr-based Identification and Southern Blottimentioning

confidence: 99%

A convenient, rapid and efficient method for establishing transgenic lines of Brassica napus

Zhang

Liu

et al. 2020

Plant Methods

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“…Nevertheless, it is plausible that the entire field of PLN functional research may have taken a different route, were the details of the genomic location of rather numerous PLN transgenes addressed early in their respective projects. Today not only the techniques of transgene characterization (review, (Stefano et al 2016)) have become substantially more accessible and affordable, but also the results of genomic mapping can be compared to a refined genome sequence and its functional annotation. They may immediately alert the researcher of any possible adverse genomic effects before physiological or behavioral studies may begin.…”

Section: Discussionmentioning

confidence: 99%

Insertional mutagenesis in PLN^R9Ctransgenic mouse: a case report and a review of the literature on cardiac-specific transgenic expression

Kraev

2016

Preprint

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A mouse line with heterozygous transgenic expression of phospholamban carrying a substitution of cysteine for arginine 9 (PLNR9C) under the control of α-myosin heavy chain (αMHC) promoter features dilated cardiomyopathy, heart failure and premature death. In this line the transgenic array of 13 PLNR9C expression cassettes, arranged in a head-to-tail tandem orientation, has integrated into the homologous genomic site, the bi-directional promoter of the αMHC (Myh6) gene and the gene for the regulatory non-coding RNA Myheart (Mhrt), both of which are involved in the execution of the α/β MHC switch during cardiac development and pathology. PLNR9C overexpression is evident at the age of 1 month but declines dramatically along with a less pronounced concomitant decline of the resident PLN expression, until the animals die. Expression of the non-coding RNA Mhrt in PLNR9C mice also exhibits a profound deregulation, despite the presence of the second, intact allele. Hence the mouse strain does not faithfully model a human PLNR9C heterozygote, wherein both the mutant and the wildtype PLN alleles have, in all likelihood, the same temporal expression profile. The intricate regulatory circuit of the α/β MHC switch, involving the non-coding RNA Mhrt, was described in detail only recently, and since publications about αMHC-driven transgenes rarely contain the definition of the transgene integration site or temporal expression profile, it is suggested that some of the pathological phenomena attributed to expression of αMHC-driven transgenes may have an alternative explanation.

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“…For Southern blotting analysis in transgenic rapeseed plants, total genomic DNA was extracted using the standard CTAB method to obtain a great amount of DNA [39]. 30 μg DNA from each sample was digested with EcoR I, which was flanking the DsRed gene in E1, E2…”

Section: Dna Extraction Pcr-based Identification and Southern Blottimentioning

confidence: 99%

A Novel Method for High-efficiently Obtaining the Transgenic Lines by combining a convenient, rapid and efficient Visual Screening method and an efficient genetic transformation System in B. napus

Zhang

Liu

et al. 2019

Preprint

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Background Brassica napus is an important oilseed crop which offer a considerable amount for global vegetable oil production. The establishment of an efficient genetic transformation system with a convenient transgenic-positive screening method is of great importance for gene functional analysis and molecular breeding. However, to our knowledge, there are few such aforementioned systems available for efficient application in B. napus .Results Based on the well-established genetic transformation system in Brassica napus , five vectors companied with red fluorescence protein from Discosoma sp. ( DsRed ) were constructed and transformed via agrobacterium -mediated hypocotyl transformation. The average of 59.1% tissues were marked with red fluorescence by visual screening method in tissue culture medium, and 96.1% of which were amplified with the exogenous genes in eight different rapeseed varieties on average. In addition, the final transgenic-positive efficiency of the rooted plantlets was up to 90.7% from the red fluorescence marked tissues, which was much higher than that of the previous reports. Besides, DsRed also could be applicable to the seedlings, including of seed coats, roots, hypocotyls and cotyledons during seed germination. These results indicate that the high-efficiency genetic transformation system combining with the transgenic-positive visual screening method presented here will help us to efficiently and conveniently obtain transgenic-positive rapeseed plantlets.Conclusion A novel method was developed for rapidly, conveniently and high-efficiently obtaining the transgenic lines, which would help to obtain the higher proportion of transgenic positive regenerated plantlets, thereby avoiding the long period plant regeneration. This will benefit the gene functional study especially in high throughput molecular biology research.

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Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants

Cited by 25 publications

References 29 publications

A convenient, rapid and efficient method for establishing transgenic lines of Brassica napus

A convenient, rapid and efficient method for establishing transgenic lines of Brassica napus

Insertional mutagenesis in PLN^R9Ctransgenic mouse: a case report and a review of the literature on cardiac-specific transgenic expression

A Novel Method for High-efficiently Obtaining the Transgenic Lines by combining a convenient, rapid and efficient Visual Screening method and an efficient genetic transformation System in B. napus

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Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants

Cited by 25 publications

References 29 publications

A convenient, rapid and efficient method for establishing transgenic lines of Brassica napus

A convenient, rapid and efficient method for establishing transgenic lines of Brassica napus

Insertional mutagenesis in PLNR9Ctransgenic mouse: a case report and a review of the literature on cardiac-specific transgenic expression

A Novel Method for High-efficiently Obtaining the Transgenic Lines by combining a convenient, rapid and efficient Visual Screening method and an efficient genetic transformation System in B. napus

Contact Info

Product

Resources

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Insertional mutagenesis in PLN^R9Ctransgenic mouse: a case report and a review of the literature on cardiac-specific transgenic expression