plasmids carrying phic31 integrase  (Addgene inc)

Journal: G3: Genes | Genomes | Genetics

Article Title: Codon optimality influences homeostatic gene expression in zebrafish

doi: 10.1093/g3journal/jkae247

Figure Lengend Snippet: Codon optimality shapes homeostatic gene expression in zebrafish. a) Schematic illustration of the conceptual effect of codon optimality. An increased percentage of optimal codons are correlated with higher mRNA stability and increased translation efficiency. A greater portion of nonoptimal codons is associated with decreased mRNA stability and lower translation efficiency. b) Diagram depicting the reporter constructs inserted into the zebrafish genome. Each reporter is made up of the same nucleotide sequence apart from a single base insertion after the P2A in the optimal version. This mutation causes a frameshift that results in a change in optimality from being overall nonoptimal to being overall optimal, relative to the endogenous transcriptome. c) Histogram showing the relative optimality vs the number of transcripts for the zebrafish transcriptome. The optimality of the frameshift reporters is indicated, with the optimal scoring more optimal than 88% of endogenous gene and the nonoptimal just above the lowest quartile at 26%. The dotted gray line represents the mean optimality of the transcriptome ( Diez et al . 2022 ). d) Schematic of the generation of transgenic zebrafish. Injection of attB -containing frameshift constructs with phiC31 integrase mRNA into embryos harboring a genomic attP landing site results in mosaic F0 progeny. Upon outcrossing to wild-type animals, F1 generation frameshift optimal, nonoptimal, and mCherry control are produced. Subsequently, the mCherry control line is crossed with each of the frameshift lines to create dual positive F2 offspring. e) Bar plot displaying the phenotypic frequency of GFP and mCherry expression in the frameshift optimal/mCherry, frameshift nonoptimal/mCherry, and mCherry control lines upon outcrossing to wild-type fish. The violet bars show the counts from individual clutches and the numbers describe total larvae counted. f) Schematic showing how homozygous mCherry animals were generated to use a copy number control. Relative mCherry expression of each line is shown in the bar plot. F2 generation frameshift optimal/mCherry fish were in-crossed, producing clutches containing 25% homozygous mCherry progeny. Genomic DNA from frameshift optimal, frameshift nonoptimal/mCherry, and homozygous mCherry fish was assayed via qPCR and normalized by housekeeping gene eef1a1|1 . g) Bar plot showing the relative mRNA levels of GFP and mCherry in 8 dpf larvae. Frameshift optimal samples showed a GFP fold change of 4.6× compared with the frameshift nonoptimal samples, while there was no change in mCherry between frameshift optimal and frameshift nonoptimal fish. h) Box and whisker plot of fluorescence intensity measured in the trunk of 8 dpf larvae (the box indicates the IQR, the whiskers show the range of values that are within 1.5×IQR, and a horizontal line indicates the mean). i) Fluorescent images of 8 dpf larvae (right) and schematic indicating the region shown in the images and the visual adjustment used (left). The larger images are shown with brightness and contrast optimized for the frameshift optimal/mCherry line, while the smaller inset is the same image adjusted for the frameshift nonoptimal/mCherry. Dotted white lines outline the perimeter of images too dark to see. Scale bar = 150 mm. For all the pannels: n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001 via Welch's 2-tailed t -test.

Article Snippet: Plasmids carrying phiC31 integrase (pcDNA3.1 phiC31, addgene #68310) were linearized with PvuII_HF (New England Biolabs) prior to in vitro transcription using the mMESSAGE mMACHINE Kit, following manufacturer's protocols. mRNA was quantified using Qubit Fluorometric Quantification.

Techniques: Gene Expression, Construct, Sequencing, Mutagenesis, Transgenic Assay, Injection, Control, Produced, Expressing, Generated, Whisker Assay, Fluorescence