Frokjaer-Jensen C et al. (2009) Nat Neurosci "Calcium: an insignificant thing."

Frokjaer-Jensen C, Jorgensen EM

[Nat Neurosci, 2009]

Fusion of synaptic vesicles in response to calcium influx requires precise localization of voltage-gated calcium channels. A study in this issue of Nature Neuroscience identifies a previously uncharacterized protein that mediates trafficking of CaV2 calcium channels in C. elegans.

Frokjaer-Jensen C (2013) Genetics "Exciting prospects for precise engineering of Caenorhabditis elegans genomes with CRISPR/Cas9."

Frokjaer-Jensen C

[Genetics, 2013]

With remarkable speed, the CRISPR-Cas9 nuclease has become the genome-editing tool of choice for essentially all genetically tractable organisms. Targeting specific DNA sequences is conceptually simple because the Cas9 nuclease can be guided by a single, short RNA (sgRNA) to introduce double-strand DNA breaks (DSBs) at precise locations. Here I contrast and highlight protocols recently developed by eight different research groups, six of which are published in GENETICS, to modify the Caenorhabditis elegans genome using CRISPR/Cas9. This reverse engineering tool levels the playing field for experimental geneticists.

Frokjaer-Jensen C et al. (2012) Nat Methods "Improved Mos1-mediated transgenesis in C. elegans."

Ailion M, Davis MW, Frokjaer-Jensen C, Jorgensen EM

[Nat Methods, 2012]

Frokjaer-Jensen C (2015) Methods Mol Biol "Transposon-Assisted Genetic Engineering with Mos1-Mediated Single-Copy Insertion (MosSCI)."

Frokjaer-Jensen C

[Methods Mol Biol, 2015]

Transgenesis in model organisms is necessary to determine the function, expression, and subcellular localization of gene products. In Caenorhabditis elegans, injected DNA can be propagated as multicopy extrachromosomal arrays but transgenes in arrays are mosaic, over-expressed in some tissues and silenced in the germline. Here, a method to insert a transgene into a specific genomic location called Mos1-mediated single-copy insertion (MosSCI) is described. Single-copy insertion allows transgene expression at levels that approximate endogenous gene expression as well as expression in the germline.

Frokjaer-Jensen, C et al. (2011) International Worm Meeting "MosSCI: Improved efficiency and new insertion sites."

Frokjaer-Jensen, C, Jorgensen, EM, Davis, MW, Ailion, M

[International Worm Meeting, 2011]

Mos1 mediated Single Copy Insertion (MosSCI) is a method to insert a single copy of exogenous DNA into a predetermined chromosomal locationA. MosSCI works by excising a Mos1 transposon; repair inserts the transgene together with a positive selection marker (unc-119) in place of the Mos1 element. Insertions are generated by injection and are identified by the lack of fluorescent co-injection markers. The inserted DNA is stable, is expressed at approximately endogenous levels and can be expressed in the germline. To expand the utility of the technique we have generated additional insertion sites, significantly improved the insertion efficiency and developed a selection marker to kill animals with extrachromosomal arrays. Insertion sites: We have generated 4x outcrossed insertion strains for a total of seven sites on Chr. I, II, IV and X. For all insertion sites, we have made regular cloning vectors and three-way Gateway compatible vectors. We have generated bright GFP insertions into all sites to validate expression from each site and for use as balancer strains. Insertion efficiency: We exclusively use direct injection to generate insertions. We have tested modifications to the helper plasmid supplying Mos1 transposase (hyperactive mutations and different promoters) and to the injection conditions (decreased DNA concentrations). We have developed a protocol that results in few extrachromosomal arrays and insertions in 60% of the injected animals. Selection against array: One of the main time constraints of generating MosSCI insertions is the screening process. Modifications to the injection conditions result in fewer arrays but do not eliminate them altogether. To further facilitate the screen for inserts we have generated a plasmid that encodes a heat-shock inducible toxin. By including the plasmid in the injection mix all animals with an extrachromosomal array can be killed by simple heat-shock. The method is fast (a couple of hours) and it greatly improves the ease of isolating insertions; in the absence of heat-shock the plasmid only very moderately decreases the efficiency of rescue. Altogether, the improvements should significantly expand the usefulness and ease of generating transgene insertions by MosSCI. (A) Frokjaer-Jensen et al. (2008).

Frokjaer-Jensen C et al. (2008) Nat Genet "Single-copy insertion of transgenes in Caenorhabditis elegans."

Wayne Davis M, Frokjaer-Jensen C, Thummel JM, Hopkins CE, Newman BJ, Olesen SP, Jorgensen EM, Grunnet M

[Nat Genet, 2008]

At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.

Frokjaer-Jensen C et al. (2008) PLoS One "Ammonium-acetate is sensed by gustatory and olfactory neurons in Caenorhabditis elegans."

Lockery SR, Ailion M, Frokjaer-Jensen C

[PLoS One, 2008]

BACKGROUND: Caenorhabditis elegans chemosensation has been successfully studied using behavioral assays that treat detection of volatile and water soluble chemicals as separate senses, analogous to smell and taste. However, considerable ambiguity has been associated with the attractive properties of the compound ammonium-acetate (NH(4)Ac). NH(4)Ac has been used in behavioral assays both as a chemosensory neutral compound and as an attractant. METHODOLOGY/MAIN FINDINGS: Here we show that over a range of concentrations NH(4)Ac can be detected both as a water soluble attractant and as an odorant, and that ammonia and acetic acid individually act as olfactory attractants. We use genetic analysis to show that NaCl and NH(4)Ac sensation are mediated by separate pathways and that ammonium sensation depends on the cyclic nucleotide gated ion channel TAX-2/TAX-4, but acetate sensation does not. Furthermore we show that sodium-acetate (NaAc) and ammonium-chloride (NH(4)Cl) are not detected as Na(+) and Cl(-) specific stimuli, respectively. CONCLUSIONS/SIGNIFICANCE: These findings clarify the behavioral response of C. elegans to NH(4)Ac. The results should have an impact on the design and interpretation of chemosensory experiments studying detection and adaptation to soluble compounds in the nematode Caenorhabditis elegans.

Frokjaer-Jensen, C. et al. (2013) International Worm Meeting "Periodic A/T rich DNA structures promote germline expression."

Davis, M.W., Jorgensen, E.M., Frokjaer-Jensen, C.

[International Worm Meeting, 2013]

Organisms face the challenge of distinguishing their own DNA from foreign DNA such as retrotransposons and DNA transposons. This is of particular concern in the germline, where any deleterious effect of transposition can affect all subsequent generations. Small RNAs (e.g. piRNAs) play an important role in identifying and silencing foreign DNA. Here we test a complementary model for silencing transposons in the germline proposed by Fire et al. (2006). In this model, stretches of mostly intronic DNA containing Periodic A/T Clusters (PATCs) identify endogenous genes and promote germline expression by preventing heterochromatic silencing. PATCs are enriched in genes expressed in the germline and residing on autosomal arms, which are strongly enriched for repressive chromatin marks. Do germ cells silence foreign DNA without PATCs by aggressive epigenetic silencing over large genomic regions? We have tested this model by inserting and monitoring germline-expressed transgenes at random genomic locations inside a Mos1 transposon. A transgene without PATCs (Pdpy-30:GFP:H2B) is silenced at all locations outside the central region of autosomes. In contrast, a transgene with PATCs in the promoter (Ppie-1:GFP:H2B) is expressed from a much broader region of the genome: autosome centers, some expression from the arms and a few from the tip of Chr. X. This position based silencing is reproducible; targeted insertions of a Ppie-1 transgene into seven MosSCI sites along Chr. V behave similarly. An endogenous gene (Psmu-1:smu-1:GFP) with many PATCs is highly resistant to silencing and expressed from essentially all genomic locations, including the entire X chromosome. Does this type of silencing occur naturally and protect worms from foreign DNA? We show that a class of germline specific retrotransposons, Cer1, follow this pattern of silencing by identifying their genomic locations in natural C. elegans isolates. Silenced Cer1 insertions are all integrated into the arms of autosomes or the X-chromosome, whereas most strains with an active Cer1 have at least one copy in the center of an autosome. We think it is likely that PATCs promote germline expression and contribute to distinguishing endogenous genes from foreign DNA.

Frokjaer-Jensen C et al. (2010) Nat Methods "Targeted gene deletions in C. elegans using transposon excision."

Prestgard-Duke M, Jorgensen EM, Hollopeter G, Harris TW, Nix P, Taylor J, Davis MW, Moerman DG, Bastiani M, Lofgren R, Frokjaer-Jensen C

[Nat Methods, 2010]

We developed a method, MosDEL, to generate targeted knockouts of genes in Caenorhabditis elegans by injection. We generated a double-strand break by mobilizing a Mos1 transposon adjacent to the region to be deleted; the double-stranded break is repaired using injected DNA as a template. Repair can delete up to 25 kb of DNA and simultaneously insert a positive selection marker.

Frokjaer-Jensen, C et al. (2013) International Worm Meeting "MiniMos and Universal MosSCI sites - new methods for C. elegans transgenesis."

Liu, X, Dupuy, D, Jorgensen, EM, Pozniakovski, A, Flibotte, S, Davis, MW, Rebora, K, Kim, SK, Moerman, DG, Taylor, J, Sarov, M, Frokjaer-Jensen, C

[International Worm Meeting, 2013]

We have developed two new methods for stable insertion of single-copy transgenes into the C. elegans genome. First, we find that a modified Mos1 element (MiniMos) can efficiently insert transgenes (60% efficiency). The transposon can carry 45kb fosmids with high fidelity; all analyzed fosmid insertions were full length by CGH. The MiniMos element can be used with genetic (unc-119, unc-18) and antibiotic selections (NeoR and PuroR) in N2, C. briggsae and natural C. elegans isolates. From insertions of Ppie-1:GFP we observe germline expression in 47% of animals. The non-fluorescent lines appear to mainly be silenced by position effects (e.g. X chromosome inactivation). Although we cannot rule out RNA epigenetic (RNAe) silencing, several lines crossed into mut-7 were not re-activated. Second, we have developed a set of universal MosSCI insertion sites. We found that MiniMos can carry a wild-type Mos1 element and have used this to insert the ttTi5605 Mos1 element and flanking genomic sequence into dozens of sites in the genome. These create a set of universal MosSCI landing sites that are compatible with a single targeting vector (e.g. pCFJ150). The insertion sites are flanked by a co-insertion marker (NeoR or Pmyo-2:GFP ) that can be followed in crosses. We have characterized a standard insertion site on each autosome that allows robust germline expression. We have used these tools to determine somatic and germline position effects. In general, we see that the center of autosomes permit robust expression whereas insertions into autosomal arms or Chr. X are frequently partially or fully silenced. To quantify silencing, we have generated a set of >150 bright fluorescent insertions at identified locations across the genome and quantified expression of a subset by automated imaging techniques (at cellular resolution in L1 or by flow-cytometry) .