scholarly journals Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays

Plant Methods ◽  
2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Snigdha Poddar ◽  
Jaclyn Tanaka ◽  
Jamie H. D. Cate ◽  
Brian Staskawicz ◽  
Myeong-Je Cho
Keyword(s):  
Gene Expression ◽  
Suspension Culture ◽  
Genome Editing ◽  
Time Course ◽  
Transient Gene Expression ◽  
Protoplast Isolation ◽  
Transient Transfection ◽  
Study Gene Expression ◽  
Rice Calli

Abstract Background An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Results Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. Conclusion The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.

scholarly journals Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays

2020 ◽  
Author(s):  
Snigdha Poddar ◽  
Jaclyn Tanaka ◽  
Jamie H. D. Cate ◽  
Brian Staskawicz ◽  
Myeong-Je Cho
Keyword(s):  
Gene Expression ◽  
Suspension Culture ◽  
Genome Editing ◽  
Time Course ◽  
Transient Gene Expression ◽  
Protoplast Isolation ◽  
Transient Transfection ◽  
Study Gene Expression ◽  
Rice Calli

AbstractAn efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Here we report a protocol for isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and prior to transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.

scholarly journals Efficient Isolation of Protoplasts from Rice Calli With Pause Points and its Application in Transient Gene Expression and Genome Editing Assays

2020 ◽  
Author(s):  
Snigdha Poddar ◽  
Jaclyn Tanaka ◽  
Jamie H. D. Cate ◽  
Brian Staskawicz ◽  
Myeong-Je Cho
Keyword(s):  
Gene Expression ◽  
Suspension Culture ◽  
Genome Editing ◽  
Time Course ◽  
Transient Gene Expression ◽  
Protoplast Isolation ◽  
Transient Transfection ◽  
Study Gene Expression ◽  
Rice Calli

Abstract Background: An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Results: Here we report a protocol for isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and prior to transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. Conclusion: The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.

scholarly journals Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays

2020 ◽  
Author(s):  
Snigdha Poddar ◽  
Jaclyn Tanaka ◽  
Jamie H. D. Cate ◽  
Brian Staskawicz ◽  
Myeong-Je Cho
Keyword(s):  
Gene Expression ◽  
Suspension Culture ◽  
Genome Editing ◽  
Time Course ◽  
Transient Gene Expression ◽  
Protoplast Isolation ◽  
Transient Transfection ◽  
Study Gene Expression ◽  
Rice Calli

Abstract Background: An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Results: Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. Conclusion: The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.

scholarly journals Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2

2022 ◽  
Vol 23 (2) ◽  
pp. 837
Author(s):  
Sudip Biswas ◽  
Nancy J. Wahl ◽  
Michael J. Thomson ◽  
John M. Cason ◽  
Bill F. McCutchen ◽  
...  
Keyword(s):  
Genome Editing ◽  
New Technologies ◽  
Fluorescent Protein ◽  
Peanut Butter ◽  
Processing System ◽  
Protoplast Isolation ◽  
Sequencing Analysis ◽  
Ara H 2

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

scholarly journals Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing

2020 ◽  
Vol 21 (4) ◽  
pp. 1380 ◽  
Author(s):  
Giovanni Pasquini ◽  
Virginia Cora ◽  
Anka Swiersy ◽  
Kevin Achberger ◽  
Lena Antkowiak ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Time Course ◽  
Sequencing Analysis ◽  
End Joining ◽  
Dsb Repair ◽  
Repair Gene ◽  
Repair Pathways

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.

scholarly journals Capturing and Understanding the Dynamics and Heterogeneity of Gene Expression in the Living Cell

2020 ◽  
Vol 21 (21) ◽  
pp. 8278
Author(s):  
Amparo Pascual-Ahuir ◽  
Josep Fita-Torró ◽  
Markus Proft
Keyword(s):  
Gene Expression ◽  
Regulation Of Gene Expression ◽  
Transient Gene Expression ◽  
Cell Physiology ◽  
Extrinsic Factors ◽  
Intrinsic Factors ◽  
Gene Expression Dynamics ◽  
Transcriptional Memory ◽  
External Stimuli

The regulation of gene expression is a fundamental process enabling cells to respond to internal and external stimuli or to execute developmental programs. Changes in gene expression are highly dynamic and depend on many intrinsic and extrinsic factors. In this review, we highlight the dynamic nature of transient gene expression changes to better understand cell physiology and development in general. We will start by comparing recent in vivo procedures to capture gene expression in real time. Intrinsic factors modulating gene expression dynamics will then be discussed, focusing on chromatin modifications. Furthermore, we will dissect how cell physiology or age impacts on dynamic gene regulation and especially discuss molecular insights into acquired transcriptional memory. Finally, this review will give an update on the mechanisms of heterogeneous gene expression among genetically identical individual cells. We will mainly focus on state-of-the-art developments in the yeast model but also cover higher eukaryotic systems.

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