New Reagents Deposited with Addgene
Feb 8, 2017
We have deposited several plasmids with C. elegans optimized fluorophores with and without PATC. In our hands, including PATCs significantly diminishes epigenetic silencing in the germline but has no obvious positive or negative effect on somatic expression. The plasmids are described here and can be accessed at Addgene here.
Paper on Periodic An/Tn Clusters (PATCs) published
July 6, 2016
Our paper on epigenetic silencing of transgenes in the C. elegans germline just came out.
- Periodic, non-coding DNA can prevent transgenes from stochastic silencing in germline
- Non-coding content of genes is shaped by genomic context and heterochromatin domains
- Conditioning of active DNA may allow cells to distinguish foreign from host genes
The worm genome contains a prevalent (~10% of the genomic sequence) non-coding DNA feature called PATCs, which is highly enriched “around” genes expressed in the germline. PATCs are highly enriched in genes that reside in repressive chromatin environments, suggesting that this non-coding protects endogenous genes from epigenetic silencing. We propose that PATCs may also play a role in a basic cellular immune system to detect foreign DNA. In this model, germ cells use this abundant non-coding DNA as a signature to identify endogenous genes and to silence foreign DNA (transposons, retroviruses, transgenes) that lack PATCs by default. In line with this, we find that transgenes with PATCs are much less frequently silenced in the germline from permissive and repressive chromatin domains.
Fluorescent markers strains with GFP insertions.
In collaboration with Ann Rougvie at the CGC, we have generated a set of strains carrying GFP markers at defined genomic locations. The strains are available from the CGC and you can search for the strains based on chromosome, fluorescent marker, and the selection marker (see Fluorescent marker strains). The fluorescence is visible on a fluorescence dissection microscope and the strains can be used to build double mutants in crosses or for outcrossing mutations isolated in genetic screens. The strains complement the previously published set of strains with insertions of tdTomato and brings the total to 294 strains.
In addition, the CGC is generating fluorescently marked versions of some of the classic balancer strains by targeted integration using Cas9. We have generated a marked MT1000 strain (unc-5(e53)/nT1 IV; dpy-11(e224)/nT1 V) by inserting Peft-3:GFP into Chr. V at 2.8 MB. The strain can be used as a balancer even though the cytosolic GFP is somewhat dim. We have therefore switched to inserting a brighter Peft-3:tdTomato(NLS) marker.
Move to Andrew Fire’s lab at Stanford
Sept 16, 2014
I have just recently moved to Andrew Fire‘s lab at Stanford! In addition to developing techniques to engineer C. elegans, I will also work on understanding how cells in the germline identify foreign and endogenous genes as a postdoc in Andy’s lab (technically, a “visiting instructor”).
Thanks to Erik and everyone in the lab for a fantastic time at University of Utah!
Antibiotic selection protocol
July 2, 2014
I have added a page with the protocol we use in Erik’s lab for antibiotic selection.
Variable efficiency of peel-1 negative selection
June 17, 2014
For no obvious reason, I have recently observed large variability in the efficiency in using the peel-1 negative selection to kill animals with extra-chromosomal arrays. When I initially characterized the negative selection (Frøkjær-Jensen et al., 2010) it was very efficient at killing essentially all animals with extra-chromosomal arrays. For a long time, I and others in Erik’s lab picked animals with mosSCI or miniMos insertions based on surviving the heat-shock and found essentially no false positives (by a secondary screen for mCherry co-injection markers). However for the past two summers, the negative selection has become very inefficient even when using the same injection mixes that previously worked. It is very strange but it coincides closely with the installation of a new evaporative cooling system in the building and the summer months. I have performed a number of experiments to enhance the efficiency of negative selection:
– Using the genomic peel-1 fragment instead of cDNA.
– Heat-shock in water bath instead of in an air incubator.
– Higher temperature heat-shock (1 hour at 37°C instead of 34°C).
– Doubling the concentration of the peel-1 plasmid (pMA122)
None of these changes improved the negative selection.
I apologize if others are observing similar variability. This was not something I observed when originally characterizing the negative selection. And if anyone has figured out how to reduce the variability (other than wait for fall and winter) then please send me an email. Or write up a paper on how “A sperm-delivered toxin can determine the seasons in the short-lived model organism C. elegans“…