Visualization and Quantification of the Dynamics of Actin Filaments in Arabidopsis Pollen Tubes.

The actin cytoskeleton plays an essential role in the regulation of polarized pollen tube growth, and its functions are dictated by its spatial organization and dynamics. Here we describe an assay to monitor the dynamics of actin filaments decorated with Lifeact-mEGFP in Arabidopsis pollen tubes using spinning disk confocal microscopy and measuring the parameters associated with their dynamics. The method allows us to assess the dynamics of actin filaments in growing Arabidopsis pollen tubes.

Correction of monomeric enhanced green fluorescent protein (mEGFP) gene by short 5'-tailed duplexes.

Mutations of important genes elicit various disorders, including cancer. Recently, a new version of a 5'-tailed duplex (short TD), consisting of a ∼100-base editor strand containing the wild-type sequence and a ∼35-base assistant strand, was shown to correct a base substitution mutation in a target gene in human cells. In that previous study, the target was the copepod green fluorescent protein (copGFP) gene. To examine the usefulness of the short TD, we performed gene correction experiments using a mutant form of the monomeric enhanced Aequorea victoria green fluorescent protein (mEGFP) gene containing a TAC to CAC mutation in codon 75 (corresponding to the tyrosine to histidine substitution in the chromophore). The short TDs with the wild-type sequence efficiently corrected the inactivated gene in human U2OS cells. These results indicated that the short TDs are effective for gene editing.

Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.

We describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.