Here, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficient
The lines provide proof‐of‐principle for the application of
This content will become publicly available on April 15, 2024
- Award ID(s):
- 2203311
- NSF-PAR ID:
- 10421568
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Date Published:
- Journal Name:
- Development
- Volume:
- 150
- Issue:
- 8
- ISSN:
- 0950-1991
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Background Astyanax mexicanus Results Tol2 system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter expresses enhanced green fluorescent protein ubiquitously throughout development in a surface population ofAstyanax . To define specific cell‐types, a Cntnap2‐mCherry construct labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish.Conclusion Tol2 transgenic technology inA. mexicanus -
While piggyBac transposon-based transgenesis is widely used in various emerging model organisms, its relatively low transposition rate in butterflies and moths has hindered its use for routine genetic transformation in Lepidoptera. Here, we tested the suitability of a codon-optimized hyperactive piggyBac transposase ( hyPBase ) in mRNA form to deliver and integrate transgenic cassettes into the genome of the pantry moth Plodia interpunctella . Co-injection of hyPBase mRNA with donor plasmids successfully integrated 1.5–4.4 kb expression cassettes driving the fluorescent markers EGFP, DsRed, or EYFP in eyes and glia with the 3xP3 promoter. Somatic integration and expression of the transgene in the G 0 injected generation was detectable from 72-h embryos and onward in larvae, pupae and adults carrying a recessive white-eyed mutation. Overall, 2.5% of injected eggs survived into transgene-bearing adults with mosaic fluorescence. Subsequent outcrossing of fluorescent G 0 founders transmitted single-insertion copies of 3xP3::EGFP and 3xP3::EYFP and generated stable isogenic lines. Random in-crossing of a small cohort of G 0 founders expressing 3xP3::DsRed yielded a stable transgenic line segregating for more than one transgene insertion site. We discuss how hyPBase can be used to generate stable transgenic resources in Plodia and other moths.more » « less
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The receptor tyrosine kinase Ret plays a critical role in regulating enteric nervous system (ENS) development. Ret is important for proliferation, migration, and survival of enteric progenitor cells (EPCs). Ret also promotes neuronal fate, but its role during neuronal differentiation and in the adult ENS is less well understood. Inactivating RET mutations are associated with ENS diseases, e.g., Hirschsprung Disease, in which distal bowel lacks ENS cells. Zebrafish is an established model system for studying ENS development and modeling human ENS diseases. One advantage of the zebrafish model system is that their embryos are transparent, allowing visualization of developmental phenotypes in live animals. However, we lack tools to monitor Ret expression in live zebrafish. Here, we developed a new BAC transgenic line that expresses GFP under the ret promoter. We find that EPCs and the majority of ENS neurons express ret:GFP during ENS development. In the adult ENS, GFP+ neurons are equally present in females and males. In homozygous mutants of ret and sox10—another important ENS developmental regulator gene—GFP+ ENS cells are absent. In summary, we characterize a ret:GFP transgenic line as a new tool to visualize and study the Ret signaling pathway from early development through adulthood.more » « less
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null (Ed.)Cell cultures are effective supplemental models to study specific biochemical pathways used for environmental adaption in animals. They enable isolation from system influence and facilitate control the extracellular environment. For work focusing on fish species many representative cell lines now exist, including a tilapia brain cell line (OmB) developed in our lab. CRISPR/Cas9 gene editing is an additional tool aiding these studies by allowing manipulation of specific genetic loci and evaluating their causal relationship between phenotypes of interest. However, established CRISPR/Cas9 gene targeting tools and methods often have not functioned as efficiently in fish cells as seen in other animal cell models such as mammalian cell lines, consistent with our initial attempts to apply CRISPR/Cas9 in OmB cells that failed to indicate genomic alteration at the targeted sites. Poor expression of heterologous promoters in OmB cells was hypothesized to be a primary cause for this occurrence so we constructed a custom plasmid vector based system utilizing tilapia endogenous promoters (EF1 alpha to express Cas9 and a U6 to express gRNAs). This system demonstrated substantial editing of most target sites attempted with mutational efficiency as high 80%. This work specifically highlighted the importance of phylogenetic proximity in selection of a polymerase III promoter for gRNA expression as commonly used interspecies U6 promoters (human and zebrafish) yielded no detectable gene editing when applied in this system with a common gRNA target sequence. These new tools will allow generation of knockout cell lines for gene targeting studies in tilapia and other phylogenetically close fish species.more » « less
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Abstract Introduction Melatonin levels are partially driven by the parenchyma volume of the pineal gland. Low urinary levels of 6‐sulfatoxymelatonin have been associated with increased risk of advanced prostate cancer, but the relationship between pineal gland volume and composition and prostate cancer risk has not been examined.
Materials and Methods We utilized data from 864 men from the AGES‐Reykjavik Study with complete pineal gland volumes and urinary 6‐sulfatoxymelatonin measurements. Pineal parenchyma, calcification, and cyst volumes were calculated from brain magnetic resonance imaging. Levels of 6‐sulfatoxymelatonin were assayed from prediagnostic urine samples. We calculated Pearson correlation coefficients between parenchyma volume and urinary 6‐sulfatoxymelatonin levels. We used Cox proportional hazards regression to calculate multivariable hazard ratios (HRs) and 95% confidence intervals (95% CIs) comparing prostate cancer risk across parenchyma volume tertiles and across categories factoring in parenchyma volume, gland composition, and urinary 6‐sulfatoxymelatonin level.
Results Parenchyma volume was moderately correlated with urinary 6‐sulfatoxymelatonin level (
r = .24;p < .01). There was no statistically significant association between parenchyma volume tertile and prostate cancer risk. Men with high parenchyma volume, pineal cysts and calcifications, and low urinary 6‐sulfatoxymelatonin levels had almost twice the risk of total prostate cancer as men with low parenchyma volume, no pineal calcifications or cysts, and low urinary 6‐sulfatoxymelatonin levels (HR: 1.98; 95% CI: 1.02, 3.84;p : .04).Conclusions Although parenchyma volume is not associated with prostate cancer risk, pineal gland composition and other circadian dynamics may influence risk for prostate cancer. Additional studies are needed to examine the interplay of pineal gland volume, composition, and melatonin levels on prostate cancer risk.
