Promoter cross-talk affects the inducible expression of intronic shRNAs from the tetracycline response element

Park, Seong Kyun; Hwang, Byung Joon; Kee, Yun

doi:10.1007/s13258-019-00784-z

Cited by 3 publications

(4 citation statements)

References 32 publications

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“…24 Our data build on work in other systems that have demonstrated that the arrangement of expression cassettes in a bi-cistronic transgene can affect transgene expression. When a second promoter is added, the activity of both the upstream and downstream promoter was shown to be affected in eukaryotes with lentiviral 19 and transient transfection systems, 20 and in prokaryotes with transient transfection systems. 32 Although each of these studies examined two polymerase II promoters, the two studies that compared placement of the downstream promoter in reverse compared to a forward direction found that the reverse direction enhanced expression of both promoters.…”

Section: Discussionmentioning

confidence: 99%

“…32 Although each of these studies examined two polymerase II promoters, the two studies that compared placement of the downstream promoter in reverse compared to a forward direction found that the reverse direction enhanced expression of both promoters. 20,32 How bi-cistronic constructs are affected also appears to depend on the promoter used, in addition to how they are arranged. For example, an interphotoreceptor retinoid binding protein promoter suppressed the activity of a rhodopsin kinase promoter, but only when located in the downstream not the upstream position.…”

Section: Discussionmentioning

confidence: 99%

“…Achieving optimal expression of Cas9 and gRNA is important to maximize on-target editing efficiency and minimize the required vector dose to be delivered. While the arrangement of expression cassettes within a bi-cistronic construct can affect expression of one or both of the transgenes, [19][20][21][22][23] little is known about how vector design affects Cas9 and gRNA expression in a single AAV system. Some authors have noted that the orientation of the U6-gRNA transcriptional unit within a vector can affect the efficiency of gene editing.…”

Section: Introductionmentioning

confidence: 99%

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Promoter Orientation within an AAV-CRISPR Vector Affects Cas9 Expression and Gene Editing Efficiency

et al. 2020

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Adeno-associated virus (AAV) vectors have been widely adopted for delivery of CRISPR-Cas components, especially for therapeutic gene editing. For a single vector system, both the Cas9 and guide RNA (gRNA) are encoded within a single transgene, usually from separate promoters. Careful design of this bi-cistronic construct is required due to the minimal packaging capacity of AAV. We investigated how placement of the U6 promoter expressing the gRNA on the reverse strand to SaCas9 driven by a cytomegalovirus promoter affected gene editing rates compared to placement on the forward strand. We show that orientation in the reverse direction reduces editing rates from an AAV vector due to reduced transcription of both SaCas9 and guide RNA. This effect was observed only following AAV transduction; it was not seen following plasmid transfection. These results have implications for the design of AAV-CRISPR vectors, and suggest that results from optimizing plasmid transgenes may not translate when delivered via AAV.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Promoter Orientation within an AAV-CRISPR Vector Affects Cas9 Expression and Gene Editing Efficiency

et al. 2020

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“…Anti-sense RNA probes were prepared using a digoxigenin– or fluorescein-labeling mix (Roche, Basel, Switzerland). To generate dand5 and lft2 RNA probes, 700–800 base pairs of each DNA fragment were amplified from zebrafish cDNA and cloned into pBluescript KS+ vector using oligonucleotide primers (Park et al 2019 ). The foxa3 and the myl7 probe constructs were kindly provided by Prof. Donghun Shin from the University of Pittsburgh School of Medicine, USA and Prof. Hyunju Ro from the Chungnam National University, Republic of Korea, respectively.…”

Section: Methodsmentioning

confidence: 99%

Histopathological assessment of laterality defects in zebrafish development

Chowdhury

Raslan

Lee

et al. 2021

Animal Cells and Systems

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Laterality defects during embryonic development underlie the aetiology of various clinical symptoms of neuropathological and cardiovascular disorders; however, experimental approaches to understand the underlying mechanisms are limited due to the complex organ systems of vertebrate models. Zebrafish have the ability to survive even when the heart stops beating for a while during early embryonic development and those adults with cardiac abnormalities. Therefore, we induced laterality defects and investigated the occurrence of situs solitus, situs inversus, and situs ambiguus in zebrafish development. Histopathological analysis revealed heterotaxy in both embryos and juvenile fish. Additionally, randomization of left-right asymmetry of the brain and heart in individual zebrafish embryos under artificial experimental pressure further demonstrated the advantage of transparent zebrafish embryos as an experimental tool to select or reduce the embryos with laterality defects during early embryonic development for long-term studies, including behavioural and cognitive neuroscience investigations.

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Toward Tightly Tuned Gene Expression Following Lentiviral Vector Transduction

Page

Fusil

Cosset

2020

Viruses

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Lentiviral vectors are versatile tools for gene delivery purposes. While in the earlier versions of retroviral vectors, transgene expression was controlled by the long terminal repeats (LTRs), the latter generations of vectors, including those derived from lentiviruses, incorporate internal constitutive or regulated promoters in order to regulate transgene expression. This allows to temporally and/or quantitatively control transgene expression, which is required for many applications such as for clinical applications, when transgene expression is required in specific tissues and at a specific timing. Here we review the main systems that have been developed for transgene regulated expression following lentiviral gene transfer. First, the induction of gene expression can be triggered either by external or by internal cues. Indeed, these regulated vector systems may harbor promoters inducible by exogenous stimuli, such as small molecules (e.g., antibiotics) or temperature variations, offering the possibility to tune rapidly transgene expression in case of adverse events. Second, expression can be indirectly adjusted by playing on inserted sequence copies, for instance by gene excision. Finally, synthetic networks can be developed to sense specific endogenous signals and trigger defined responses after information processing. Regulatable lentiviral vectors (LV)-mediated transgene expression systems have been widely used in basic research to uncover gene functions or to temporally reprogram cells. Clinical applications are also under development to induce therapeutic molecule secretion or to implement safety switches. Such regulatable approaches are currently focusing much attention and will benefit from the development of other technologies in order to launch autonomously controlled systems.

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Promoter cross-talk affects the inducible expression of intronic shRNAs from the tetracycline response element

Cited by 3 publications

References 32 publications

Promoter Orientation within an AAV-CRISPR Vector Affects Cas9 Expression and Gene Editing Efficiency

Promoter Orientation within an AAV-CRISPR Vector Affects Cas9 Expression and Gene Editing Efficiency

Histopathological assessment of laterality defects in zebrafish development

Toward Tightly Tuned Gene Expression Following Lentiviral Vector Transduction

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