Dataset Information

Gene Expression and Genome Editing Systems by Direct Delivery of Macromolecules Into Rice Egg Cells and Zygotes.

ABSTRACT: Polyethylene glycol calcium (PEG-Ca²⁺)-mediated transfection allows rapid and efficient examination to analyze diverse cellular functions of genes of interest. In plant cells, macromolecules, such as DNA, RNA and protein, are delivered into protoplasts derived from somatic tissues or calli via PEG-Ca²⁺ transfection. To broaden and develop the scope of investigations using plant gametes and zygotes, a procedure for direct delivery of macromolecules into these cells has recently been established using PEG-Ca²⁺ transfection. This PEG-Ca²⁺-mediated delivery into rice egg cells/zygotes consists of four microtechniques, (i) isolation of gametes, (ii) production of zygotes by electrofusion of gametes, (iii) PEG-Ca²⁺-mediated delivery of macromolecules into isolated egg cells or produced zygotes, and (iv) culture and subsequent analyses of the transfected egg cells/zygotes. Because the full protocol for microtechniques (i) and (ii) have already been reported in Toda et al., 2016 , microtechniques (iii) and (iv) are mainly described in this protocol.

SUBMITTER: Toda E

PROVIDER: S-EPMC7842353 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Gene Expression and Genome Editing Systems by Direct Delivery of Macromolecules Into Rice Egg Cells and Zygotes.

Toda Erika E Okamoto Takashi T

Bio-protocol 20200720 14

Polyethylene glycol calcium (PEG-Ca<sup>2+</sup>)-mediated transfection allows rapid and efficient examination to analyze diverse cellular functions of genes of interest. In plant cells, macromolecules, such as DNA, RNA and protein, are delivered into protoplasts derived from somatic tissues or calli via PEG-Ca<sup>2+</sup> transfection. To broaden and develop the scope of investigations using plant gametes and zygotes, a procedure for direct delivery of macromolecules into these cells has recen ...[more]

PMID: 33659352

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Project description:The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes. There are a number of studies examining the parameters used in electroporation of mouse and rat zygotes. Here, we review the electroporation conditions, timing, and success rates that have been reported for mice and rats, in addition to the few reports about livestock zygotes, specifically pigs and cattle. The introduction of editing reagents at, or soon after, fertilization can help reduce the rate of mosaicism, the presence of two of more genotypes in the cells of an individual; as can the introduction of nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic in large livestock species with long generation intervals as it can take years to obtain non-mosaic, homozygous offspring through breeding. Gene knockouts accomplished via the non-homologous end joining pathway have been more widely reported and successfully accomplished using electroporation than have gene knock-ins. Delivering large DNA plasmids into the zygote is hindered by the zona pellucida (ZP), and the majority of gene knock-ins accomplished by electroporation have been using short single stranded DNA (ssDNA) repair templates, typically less than 1 kb. The most promising approach to deliver larger donor repair templates of up to 4.9 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. However, similar to other methods used to deliver clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this approach is also associated with high levels of mosaicism. Recent developments complementing germline ablated individuals with edited germline-competent cells offer an approach to avoid mosaicism in the germline of genome edited founder lines. Even with electroporation-mediated delivery of genome editing reagents to mammalian zygotes, there remain additional chokepoints in the genome editing pipeline that currently hinder the scalable production of non-mosaic genome edited livestock.

Dataset Information

Gene Expression and Genome Editing Systems by Direct Delivery of Macromolecules Into Rice Egg Cells and Zygotes.

Publications

Gene Expression and Genome Editing Systems by Direct Delivery of Macromolecules Into Rice Egg Cells and Zygotes.

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