Elimination of Transgenic Sequences in Plants by Cre Gene Expression

Kopertekh, Lilya; Schiemann, Joachim

doi:10.5772/32943

Cited by 6 publications

(4 citation statements)

References 92 publications

(106 reference statements)

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“…These technologies have been tested for the removal of marker genes in different plant species. The recombination success ranges from 0.25% to 82% for transient expression methodologies, from 13% to 100% for inducible Cre expression, and from 19.2% to 95% for cross pollination with a Cre-expressing line (for review, see Kopertekh and Schiemann, 2012 Hare and Chua, 2002). Compared with these technologies, MSN-mediated Cre protein delivery is a straightforward method that does not require complex DNA constructs, analyses to detect unwanted DNA integration, or genetic engineering of microorganisms.…”

Section: Discussionmentioning

confidence: 99%

Mesoporous Silica Nanoparticle-Mediated Intracellular Cre Protein Delivery for Maize Genome Editing via loxP Site Excision^,

et al. 2013

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Section: Discussionmentioning

confidence: 99%

Mesoporous Silica Nanoparticle-Mediated Intracellular Cre Protein Delivery for Maize Genome Editing via loxP Site Excision^,

et al. 2013

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“…LoxP-frt fusion sequences, combining wild type loxP and frt, can provide higher recombination rates than wild type systems, when used as flanking sites for FLP or CRE recombinase (Luo et al 2007). Temporal or developmental regulation of CRE/loxP recombination decreases/eliminates side-effects of CRE protein that have been reported before (Coppoolse et al 2003;Kopertekh and Schiemann 2012). PCR-based analysis of gene activity, as performed in this study after CRE/loxP recombination, allow a much more reliable strategy for the avoidance of genetic mosaics.…”

Section: Potential Of Recombination Systems For Genetic Containment I...mentioning

confidence: 99%

Level of tissue differentiation influences the activation of a heat-inducible flower-specific system for genetic containment in poplar (Populus tremula L.)

Hoenicka¹,

Lehnhardt²,

Nunna

et al. 2015

Plant Cell Rep

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Differentiation level but not transgene copy number influenced activation of a gene containment system in poplar. Heat treatments promoted CRE gene body methylation. The flower-specific transgene deletion was confirmed. Gene flow between genetic modified trees and their wild relatives is still motive of concern. Therefore, approaches for gene containment are required. In this study, we designed a novel strategy for achieving an inducible and flower-specific transgene removal from poplar trees but still expressing the transgene in the plant body. Hence, pollen carrying transgenes could be used for breeding purposes under controlled conditions in a first phase, and in the second phase genetic modified poplars developing transgene-free pollen grains could be released. This approach is based on the recombination systems CRE/loxP and FLP/frt. Both gene constructs contained a heat-inducible CRE/loxP-based spacer sequence for in vivo assembling of the flower-specific FLP/frt system. This allowed inducible activation of gene containment. The FLP/frt system was under the regulation of a flower-specific promoter, either CGPDHC or PTD. Our results confirmed complete CRE/loxP-based in vivo assembling of the flower-specific transgene excision system after heat treatment in all cells for up to 30 % of regenerants derived from undifferentiated tissue cultures. Degradation of HSP::CRE/loxP spacer after recombination but also persistence as extrachromosomal DNA circles were detected in sub-lines obtained after heat treatments. Furthermore, heat treatment promoted methylation of the CRE gene body. A lower methylation level was detected at CpG sites in transgenic sub-lines showing complete CRE/loxP recombination and persistence of CRE/loxP spacer, compared to sub-lines with incomplete recombination. However, our results suggest that low methylation might be necessary but not sufficient for recombination. The flower-specific FLP/frt-based transgene deletion was confirmed in 6.3 % of flowers.

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“…However, reports about successful gene deletion by the system in plants are rare compared with that in animals. Although a proof‐of‐principle has been demonstrated, no practical application of the system is reported in plants (Kopertekh & Schiemann, 2012). The mechanism underlying the poor gene‐deletion performance of the recombinase system in plants remains to be elucidated (Russell et al, 1992; Arumugam et al, 2007; Chakraborti et al, 2008; Hoenicka et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

DNA methylation occurring in Cre‐expressing cells inhibits loxP recombination and silences loxP‐sandwiched genes

et al. 2021

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The low DNA recombination efficiency of site-specific recombinase systems in plants limits their application; however, the underlying mechanism is unknown.We evaluate the gene deletion performance of four recombinase systems (Cre/loxP, Flp/ FRT, KD/KDRT and B3/B3RT) in tobacco where the recombinases are under the control of germline-specific promoters.We find that the expression of these recombinases results mostly in gene silencing rather than gene deletion. Using the Cre/loxP system as a model, we reveal that the region flanked by loxP sites (floxed) is hypermethylated, which prevents floxed genes from deletion while silencing the expression of the genes. We further show CG methylation alone in the recombinase binding element of the loxP site is unable to impede gene deletion; instead, CHH methylation in the crossover region is required to inhibit loxP recombination.Our study illustrates the important role of recombinase-induced DNA methylation in the inhibition of site-specific DNA recombination and uncovers the mechanism underlying recombinase-associated gene silence in plants.

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Elimination of Transgenic Sequences in Plants by Cre Gene Expression

Cited by 6 publications

References 92 publications

Mesoporous Silica Nanoparticle-Mediated Intracellular Cre Protein Delivery for Maize Genome Editing via loxP Site Excision^,

Mesoporous Silica Nanoparticle-Mediated Intracellular Cre Protein Delivery for Maize Genome Editing via loxP Site Excision^,

Level of tissue differentiation influences the activation of a heat-inducible flower-specific system for genetic containment in poplar (Populus tremula L.)

DNA methylation occurring in Cre‐expressing cells inhibits loxP recombination and silences loxP‐sandwiched genes

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Elimination of Transgenic Sequences in Plants by Cre Gene Expression

Cited by 6 publications

References 92 publications

Mesoporous Silica Nanoparticle-Mediated Intracellular Cre Protein Delivery for Maize Genome Editing via loxP Site Excision,

Mesoporous Silica Nanoparticle-Mediated Intracellular Cre Protein Delivery for Maize Genome Editing via loxP Site Excision,

Level of tissue differentiation influences the activation of a heat-inducible flower-specific system for genetic containment in poplar (Populus tremula L.)

DNA methylation occurring in Cre‐expressing cells inhibits loxP recombination and silences loxP‐sandwiched genes

Contact Info

Product

Resources

About

Mesoporous Silica Nanoparticle-Mediated Intracellular Cre Protein Delivery for Maize Genome Editing via loxP Site Excision^,

Mesoporous Silica Nanoparticle-Mediated Intracellular Cre Protein Delivery for Maize Genome Editing via loxP Site Excision^,