Gene Stacking and Stoichiometric Expression of ER-Targeted Constructs Using “2A” Self-Cleaving Peptides

Spatola Rossi, Tatiana; Fricker, Mark; Kriechbaumer, Verena

doi:10.1007/978-1-0716-3710-4_26

Cited by 3 publications

(3 citation statements)

References 42 publications

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“…Moreover, transgenes are generally stacked in a single construct controlled by a minimum number of synthetic promoters that should not be homologous to those already used in plant genetic modification [160,167,[170][171][172]. This reduces the frequency and scale of complex segregation patterns and random integration into the plant genome [168,169]. This necessity for gene stacking, combined with the decreasing number of available native promoters, is addressed by a more broad development and application of synthetic bidirectional promoters that are relatively short and compact, enabling the expression and precise regulation of up to six-eight genes for each synthetic promoter [173][174][175][176][177]238].…”

Section: Discussionmentioning

confidence: 99%

“…One efficient solution involves using a single transgenic event to allow the coordinated expression of multiple transgenes by the minimum number of regulatory elements [160,167]. Such stacking is an optimal method of allowing the simultaneous expression of a gene set, as it integrates them into adjacent regions of the genome and reduces the probability of transgene segregation in later generations [168,169]. Multicistronic plant expression vector systems have been developed for this purpose [170][171][172].…”

Section: Objectives and General Methodology Of Synthetic Promoter Cre...mentioning

confidence: 99%

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Plant Synthetic Promoters

Szymczyk,

Majewska

2024

Applied Sciences

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This article examines the structure and functions of the plant synthetic promoters frequently used to precisely regulate complex regulatory routes. It details the composition of native promoters and their interacting proteins to provide a better understanding of the tasks associated with synthetic promoter development. The production of synthetic promoters is performed by relatively small libraries produced generally by basic molecular or genetic engineering methods such as cis-element shuffling or domain swapping. The article also describes the preparation of large-scale libraries supported by synthetic DNA fragments, directed evolution, and machine or deep-learning methodologies. The broader application of novel, synthetic promoters reduces the prevalence of homology-based gene silencing or improves the stability of transgenes. A particularly interesting group of synthetic promoters are bidirectional forms, which can enable the expression of up to eight genes by one regulatory element. The introduction and controlled expression of several genes after one transgenic event strongly decreases the frequency of such problems as complex segregation patterns and the random integration of multiple transgenes. These complications are commonly observed during the transgenic crop development enabled by traditional, multistep transformation using genetic constructs containing a single gene. As previously tested DNA promoter fragments demonstrate low complexity and homology, their abundance can be increased by using orthogonal expression systems composed of synthetic promoters and trans-factors that do not occur in nature or arise from different species. Their structure, functions, and applications are rendered in the article. Among them are presented orthogonal systems based on transcription activator-like effectors (dTALEs), synthetic dTALE activated promoters (STAPs) and dCas9-dependent artificial trans-factors (ATFs). Synthetic plant promoters are valuable tools for providing precise spatiotemporal regulation and introducing logic gates into the complex genetic traits that are important for basic research studies and their application in crop plant development. Precisely regulated metabolic routes are less prone to undesirable feedback regulation and energy waste, thus improving the efficiency of transgenic crops.

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Section: Discussionmentioning

confidence: 99%

Section: Objectives and General Methodology Of Synthetic Promoter Cre...mentioning

confidence: 99%

Plant Synthetic Promoters

Szymczyk,

Majewska

2024

Applied Sciences

View full text Add to dashboard Cite

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“…An efficient approach to addressing such an issue is the coordinated expression of multiple transgenes by the minimum number of regulatory elements in a single transgenic event [160,167]. Therefore, stacking is the optimal method of simultaneous gene set expression due to their integration into adjacent regions of the genome and reducing the probability of transgene segregation in the fortcoming generations [168,169]. Multicistronic, versatile plant expression vector systems are developed to realize such a task [170][171][172].…”

Section: Objectives and General Methodology Of Synthetic Promoter Cre...mentioning

confidence: 99%

Plant Synthetic Promoters

Szymczyk,

Majewska

2024

Preprint

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The article features the structure and functions of plant synthetic promoters frequently exercised to precisely regulate complex regulatory routes. The composition of plant native promoters together with interacting proteins is presented to provide a better understanding of tasks associated with synthetic promoter development. The production of synthetic promoters is conferred on relatively small libraries produced generally by basic molecular or genetic engineering methods such as cis-element shuffling or domain swapping. Moreover, the preparation of large-scale libraries supported by synthetic DNA fragments, directed evolution, and machine or deep learning methodologies is presented. A particularly interesting group of synthetic promoters are bidirectional forms that enable the putative expression of up to 6–8 genes by one regulatory element. The introduction and controlled expression of several genes after one transgenic event strongly decreases the frequency of such problems as complex segregation patterns and random integration of multiple transgenes. These complications are commonly observed during transgenic crop development through traditional, multistep transformation by genetic constructs containing a single gene. Another path to solving problems associated with the low complexity and homology of already tested DNA fragments is through orthogonal expression systems composed of synthetic promoters and trans-factors that do not occur in nature or arise from different species. Their structure, functions, and applications are rendered in the article.

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Suborganellar resolution imaging for the localisation of human glycosylation enzymes in tobacco Golgi bodies

McGinness,

Brooks,

Strasser

et al. 2024

Journal of Microscopy

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Plant cells are a capable system for producing economically and therapeutically important proteins for a variety of applications, and are considered a safer production system than some existing hosts such as bacteria or yeasts. However, plants do not perform protein modifications in the same manner as mammalian cells do. This can impact on protein functionality for plant‐produced human therapeutics. This obstacle can be overcome by creating a plant‐based system capable of ‘humanising’ proteins of interest resulting in a glycosylation profile of synthetic plant‐produced proteins as it would occur in mammalian systems.For this, the human glycosylation enzymes (HuGEs) involved in N‐linked glycosylation N‐acetylglucosaminyltransferase IV and V (GNTIV and GNTV), β‐1,4‐galactosyltransferase (B4GALT1), and α‐2,6‐sialyltransferase (ST6GAL) were expressed in plant cells. For these enzymes to carry out the stepwise glycosylation functions, they need to localise to late Golgi body cisternae. This was achieved by a protein targeting strategy of replacing the mammalian Golgi targeting domains (Cytoplasmic‐Transmembrane‐Stem (CTS) regions) with plant‐specific ones. Using high‐resolution and dynamic confocal microscopy, we show that GNTIV and GNTV were successfully targeted to the medial‐Golgi cisternae while ST6GAL and B4GALT1 were targeted to trans‐Golgi cisternae.Plant cells are a promising system to produce human therapeutics for example proteins used in enzyme replacement therapies. Plants can provide safer and cheaper alternatives to existing expression systems such as mammalian cell culture, bacteria or yeast. An important factor for the functionality of therapeutic proteins though are protein modifications specific to human cells. However, plants do not perform protein modifications in the same manner as human cells do. Therefore, plant cells need to be genetically modified to mimic human protein modifications patterns. The modification of importance here, is called N‐linked glycosylation and adds specific sugar molecules onto the proteins.Here we show the expression of four human glycosylation enzymes, which are required for N‐linked glycosylation, in plant cells.In addition, as these protein modifications are carried out in cells resembling a factory production line, it is important that the human glycosylation enzymes be placed in the correct cellular compartments and in the correct order. This is carried out in Golgi bodies. Golgi bodies are composed of several defined stacks termed cis‐, medial and trans‐Golgi body stacks. For correct protein function, two of these human glycosylation enzymes need to be placed in the medial‐Golgi attacks and the other two in the trans‐Golgi stacks. Using high‐resolution laser microscopy in live plant cells, we show here that the human glycosylation enzymes are sent within the cells to the correct Golgi body stacks. These are first steps to modify plant cells in order to produce human therapeutics.

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Gene Stacking and Stoichiometric Expression of ER-Targeted Constructs Using “2A” Self-Cleaving Peptides

Cited by 3 publications

References 42 publications

Plant Synthetic Promoters

Plant Synthetic Promoters

Plant Synthetic Promoters

Suborganellar resolution imaging for the localisation of human glycosylation enzymes in tobacco Golgi bodies

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