Primary Culture System for Germ Cells from Caenorhabditis elegans  Tumorous Germline Mutants

Vagasi, Alexandra S.; Rahman, Mohammad Matiur; Chaudhari, Snehal N.; Kipreos, Edward T.

doi:10.21769/bioprotoc.2424

Cited by 2 publications

(2 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The in vitro culture of isolated glp-1(gf); cki-2; daf-16 germ cells in CeM1 medium with stimulatory bacterial extract and folates was as described (Vagasi et al, 2017). Δ 7 -DA or Δ 4 -DA (both from Cayman Chemical Co.) or DMSO or ethanol carrier controls, respectively, were included in the CeM1 medium.…”

Section: Methodsmentioning

confidence: 99%

Dafachronic acid inhibits C. elegans germ cell proliferation in a DAF-12-dependent manner

Mukherjee

Chaudhari

Balachandran

et al. 2017

Developmental Biology

View full text Add to dashboard Cite

Dafachronic acid (DA) is a bile acid-like steroid hormone that regulates dauer formation, heterochrony, and lifespan in C. elegans. Here, we describe that DA is an inhibitor of C. elegans germ stem cell proliferation in adult hermaphrodites. Using a C. elegans germ cell primary culture system, we show that DA inhibits the proliferation of germ cells in vitro. Exogenous DA reduces the frequency of large tumors in adult tumorous germline mutants and decreases the proliferation of wild-type germ stem cells in adult hermaphrodites. In contrast, DA has no appreciable effect on the proliferation of larval-stage germ cells in wild type. The inhibition of adult germ cell proliferation by DA requires its canonical receptor DAF-12. Blocking DA production by inactivating the cytochrome P450 DAF-9 increases germ cell proliferation in wild-type adult hermaphrodites and the frequency of large tumors in germline tumorous mutants, suggesting that DA inhibits the rate of germ cell proliferation under normal growth conditions.

show abstract

Section: Methodsmentioning

confidence: 99%

Dafachronic acid inhibits C. elegans germ cell proliferation in a DAF-12-dependent manner

Mukherjee

Chaudhari

Balachandran

et al. 2017

Developmental Biology

View full text Add to dashboard Cite

show abstract

“…Lysing the worms is the critical step during this method since it is of crucial importance that DNA damages are not caused by the lysing process itself. We found that gentle sonification (Ultrasonic Processor, UP 100H, 2 × 20 s, in 1 mL liquid) works better for that purpose than using chemical dissipation of the cuticle (e. g. protein kinase K, pronase E, papain, Triton X-100), breaking of the cuticle with high pressure (French pressure cell press, as used for yeast cell lysis (Moore et al 2016)) or slicing worms using syringes as described for germ cell isolation (Vagasi et al 2017). Except for the gentle sonification, the other methods resulted in additional DNA damage as we detected only very low amounts of dsDNA (data not shown).…”

Section: Methods Developmentmentioning

confidence: 99%

A fast and reliable method for monitoring genomic instability in the model organism Caenorhabditis elegans

et al. 2021

View full text Add to dashboard Cite

The identification of genotoxic agents and their potential for genotoxic alterations in an organism is crucial for risk assessment and approval procedures of the chemical and pharmaceutical industry. Classically, testing strategies for DNA or chromosomal damage focus on in vitro and in vivo (mainly rodent) investigations. In cell culture systems, the alkaline unwinding (AU) assay is one of the well-established methods for detecting the percentage of double-stranded DNA (dsDNA). By establishing a reliable lysis protocol, and further optimization of the AU assay for the model organism Caenorhabditis elegans (C. elegans), we provided a new tool for genotoxicity testing in the niche between in vitro and rodent experiments. The method is intended to complement existing testing strategies by a multicellular organism, which allows higher predictability of genotoxic potential compared to in vitro cell line or bacterial investigations, before utilizing in vivo (rodent) investigations. This also allows working within the 3R concept (reduction, refinement, and replacement of animal experiments), by reducing and possibly replacing animal testing. Validation with known genotoxic agents (bleomycin (BLM) and tert-butyl hydroperoxide (tBOOH)) proved the method to be meaningful, reproducible, and feasible for high-throughput genotoxicity testing, and especially preliminary screening.

show abstract

Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants

Cited by 2 publications

References 7 publications

Dafachronic acid inhibits C. elegans germ cell proliferation in a DAF-12-dependent manner

Dafachronic acid inhibits C. elegans germ cell proliferation in a DAF-12-dependent manner

A fast and reliable method for monitoring genomic instability in the model organism Caenorhabditis elegans

Contact Info

Product

Resources

About