Frokjaer-Jensen, Christian et al. (2009) International Worm Meeting "Using Mos1 elements to modify the genome."

Newman, Blake, Jorgensen, Erik M., Olesen, Soren-Peter, Grunnet, Morten, Lofgren, Rachel, Hopkins, Christopher E., Davis, M. Wayne, Frokjaer-Jensen, Christian

[International Worm Meeting, 2009]

Transgenic C. elegans are routinely generated by injecting DNA into the germline. Injected DNA assembles into complex, yet repetitive, extrachromosomal arrays. As such, these minichromosomes are not stably inherited. Moreover, the genes are typically overexpressed in somatic cells and silenced in the germline. Alternatively, transgenes can be inserted by biolistic transformation. The bombarded DNA is stably integrated which solves many of the limitations of DNA injection. However germline expression is still infrequent. Also, DNA copy number and insertion site is essentially random. As a result, transgene expression is variable and comparison between different transgenic lines is difficult. Based on recent advances1 we developed a technique we call MosSCI2 (Mos1-mediated Single Copy Insertion) that circumvents many of these problems. Briefly, the technique functions by mobilizing a Mos1 transposon from a defined locus in non-coding DNA, thus introducing a double strand break. The break is healed by copying from an extrachromosomal template in which DNA that is homologous to the breakpoint flanks the transgene. Thereby a single copy of a transgene is inserted into the chromosomal site. We have shown that insertions can be generated either directly by injection or from stable extrachromosomal arrays. We have successfully inserted transgenes as long as 17 kb and verified that single copies are inserted at the targeted site. Inserted transgenes are expressed at endogenous levels and can be expressed in both the hermaphrodite and male germlines. We have recently expanded the technique to manipulate genomic regions adjacent to Mos1 inserts. By shifting the homology regions on the targeting vectors we have engineered defined deletions of genes adjacent to Mos1 inserts. Because unc-119 rescue is used as the positive selection marker for targeting homozygous lethal deletions are automatically balanced perfectly. The technique works efficiently by injection and deletions can be recovered in less than two weeks. 1. Robert, V. & Bessereau, J.-L. (2007) EMBO 26: 170-83 2. Frokjaer-Jensen et al., (2008). Nat. Genet. 40: 1375-83.

Frokjaer-Jensen, Christian et al. (2011) International Worm Meeting "A recombinant Mos1 transposon can carry large DNA fragments."

Taylor, J, Davis, MW, Frokjaer-Jensen, Christian, Moerman, D, Sarov, M, Jorgensen, EM, Stevenson, ZC

[International Worm Meeting, 2011]

P element-mediated transgenesis has been an important tool in Drosophila for the past 30 years. For example, P-elements have been used extensively to insert transgenes, to generate loss of function mutants and to probe the genome for regulatory regions. We have developed a minimal recombinant Mos1A transposon (miniMos) that behaves much like a P-element. Exogenous DNA placed inside a recombinant transposon is inserted into the C. elegans genome at high frequencies by simple injection (60% insertion freq.) or by heat-shock. In characterized lines, 46% inserted in intergenic regions, 43% in introns and 11% in exons (n = 54). The method is easy, very robust and works well with different mutant selection markers (unc-119 and unc-18) as well as antibiotic markers (neomycinB and puromycinC). At lower frequencies (2-14%), miniMos can carry large pieces of DNA derived from fosmids (45kb). We have verified by comparative genome hybridization (CGH) that fully intact fosmids are inserted and all 18 independent air-2::gfp fosmid insertions express visible GFP. This technique makes the generation of single-copy transgenes rapid and easy. In addition, miniMos transgenes can be used for gene-traps and for genome-wide chromatin studies. Gene trapping. The miniMos element can be used to simultaneously disrupt genes and to determine their expression pattern. We have constructed a miniMos gene-trap carrying a promoterless mCherry with an SL2 trans-splice leader sequence. In a pilot screen, we have isolated 20 lines that express mCherry in specific patterns. Each line contains a transposon inserted into a transcription unit (either exon, intron or UTR). Chromatin and gene regulation. The miniMos element can be used to probe the genome for permissive and silenced regions. Germline expressed genes are highly enriched in introns that contain phased DNAD and are virtually absent from the X chromosomeE. We can test the effects of genomic environment and phasing by inserting germline expressed transgenes. For example, a phased Ppie-1::GFP::H2B construct results in stable germline fluorescence in 67% (12 of 18) of inserts whereas similar, but non-phased Pgld-1 and Pmex-5 constructs result in much lower frequencies of fluorescence. (A) Bessereau et al., (2001) (B) Giordano-Santini et al., (2010) (C) Semple et al., (2010) (D) Fire et al., (2006) (E) Bender et al., (2006) .

Christian Frokjaer-Jensen et al. (2002) West Coast Worm Meeting "Cameleon Imaging of Calcium Transients in Cultured Mechanosensory Neurons"

Christian Frokjaer-Jensen, Katie Kindt, Massimo Hilliard, Rex Kerr, HIROSHI SUZUKI, William R Schafer

[West Coast Worm Meeting, 2002]

We have used primary C.elegans cell cultures and the genetically encoded calcium sensor cameleon to study the molecules involved in producing calcium transients in mechanosensory neurons in response to depolarization with a high K solution. The cultured neurons are amenable to genetic manipulations and to pharmacogical agents, which have been difficult to test in intact worms.

Christian Frokjaer-Jensen et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "exp-3Encodes a Small Conductance Calcium-Activated K+ Channel"

Morten Grunnet, Christian Frokjaer-Jensen, Berevan Baban, Erik Jorgensen, Soren-Peter Olesen, Lawrence Salkoff

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

The dominant mutation exp-3(n2372) results in worms strongly defective in egg-laying and enteric muscle contraction. We have mapped exp-3(n2372) to a gene encoding a putative small conductance calcium-activated K+ channel (B0399.1). The phenotypes can be suppressed by feeding worms B0399.1 RNAi and sequencing revealed a missense mutation in the intracellular linker between the predicted transmembrane regions S4 and S5. In an effort to find loss of function alleles and interacting channel subunits we conducted a suppressor screen on exp-3(n2372). We screened approximately 90,000 haploid genomes and isolated 32 suppressors, all of which were intragenic revertants. Many revertants are early stop codons or missense mutations affecting regions important for channel ion selectivity, membrane stability or calmodulin binding. Preliminary results suggest that loss of function mutants have a weak expulsion defect. A transcriptional GFP fusion to the short c-isoform of B0399.1 shows expression in the vulval muscles, anal depressor muscle, the phasmids, the harsh touch neuron PVD and at least two amphid sensory neurons. The phenotypes are consistent with a direct role of exp-3 in the vulval and anal depressor muscles. We hypothesize that the exp-3 mutation results in an inappropriately activated K+ channel leading to loss of muscle excitability. Sequence comparison between B0399.1 and mammalian homologs indicates limited overall homology, but residues mutated in the suppressor screen are highly conserved. We are interested in learning how the exp-3 mutation affects channel function and what the channel"s normal function is in worms.

Zeiser, Eva A. et al. (2009) International Worm Meeting "MosSCI and Gateway compatible toolkit for germline expression of transgenes."

Jorgensen, Erik, Ahringer, Julie, Frokjaer-Jensen, Christian, Zeiser, Eva A.

[International Worm Meeting, 2009]

Transgene silencing in the germline has hampered research in the germline and early embryo. The creation of "complex" extrachromosomal arrays using genomic DNA and then the development of technology for low copy number insertions using microparticle bombardment finally allowed germline expression of transgenes (Mello et al., 1991; Praitis et al., 2001). However, bombardment is very labor intensive and complex extrachromosomal arrays often are silenced. Recently, the Mos1 mediated Single Copy Insertion (MosSCI) method was developed to insert single copies of transgenes into defined sites in the genome of Caenorhabditis elegans (Frokjaer-Jensen et al., 2008). The technique is based on the MosTIC technique (Robert & Bessereau, 2007). Directed integration of a single copy avoids problems like overdosage, passage related loss of transgenes and locus mediated phenotypes. It also avoids extrachromosomal array related gene silencing in the germline which makes it especially attractive for studies on the gonad, and early embryos. We are using the MultiSite Gateway system to generate transgenes for germline gene expression. This method allows easy fusion of three different sequences in a defined order (5'', middle, and 3''). We have generated a set of 5'' constructs containing the germline active promoter pmex-5 (Merritt et al., 2008) on its own or fused to the sequences of GFP, EGFP, Citrine and Tag RFP-T. Similarly, we have generated a set of 3'' constructs by fusing GFP, EGFP or Citrine to the tbb-2 3''UTR (Merritt et al., 2008). Using an appropriate combination of 5'' and 3'' constructs with a middle ORF of one''s choice allows easy generation of N- or C-terminal fluorescently tagged transgenes for expression in the germline or early embryo. We also show that the hsp-16.2 and hsp-16.41 heat shock promoters can be used for inducible germline and early embryo gene expression as single copy MosSCI generated insertions.

Laine, Viviane et al. (2011) International Worm Meeting "EGL-19, UNC-36 and CCB-1 underlie voltage-dependent calcium currents in C. elegans striated muscle."

Jospin, Maelle, Laine, Viviane, Frokjaer Jensen, Christian

[International Worm Meeting, 2011]

Voltage-gated calcium channels, which play key roles in many physiological processes, are composed of a pore-forming a1 subunit associated with three auxiliary subunits. In vertebrates, the role of auxiliary subunits has mostly been studied in heterologous systems, partially because of the severe phenotypes of knockout animals. The genetic model Caenorhabditis elegans has all main types of voltage-gated calcium channels and strong loss-of-function mutations in all pore-forming and auxiliary subunits; it is therefore a useful model to investigate the roles of auxiliary subunits in their native context. By recording calcium currents from mutants of the different subunits, we molecularly dissected the voltage-dependent calcium currents in striated muscle of C. elegans. We first showed that EGL-19 is the only a1 subunit that carries calcium currents in muscle cells. We then demonstrated that the a2/d subunit UNC-36 modulates the voltage-dependence, activation kinetics and conductance of calcium currents, whereas another a2/d subunit TAG-180 has no effect. Finally, we characterized mutants of the two b subunits, CCB-1 and CCB-2. We showed that (i) locomotion is impaired in ccb-1 animals whereas it is similar to wild type for ccb-2 mutants, (ii) ccb-1 is expressed in most muscle and neuronal tissues whereas ccb-2 is restricted to a few neurons of the head and of the ventral cord, and (iii) voltage-dependence and conductance of calcium currents are severely altered in striated muscle cells of ccb-1 mutants whereas they are similar to wild type in ccb-2 mutants. Altogether these results show that EGL-19, UNC-36 and CCB-1 underlie voltage-dependent calcium currents in C. elegans striated muscle.

Artiles, Karen et al. (2021) International Worm Meeting "A tool for warp speed genetics in C. elegans"

Frokjaer-Jensen, Christian, Artiles, Karen, Fire, Andrew

[International Worm Meeting, 2021]

What if we could rapidly and reliably homozygose every locus in the C. elegans genome during genetic manipulations? What if we could combine the full nuclear information from one C. elegans strain with cytoplasmic contents from a second strain? We can do both, and it is remarkably easy! Besseling and Bringmann (2016) identified a molecular intervention for C. elegans in which premature segregation of maternal and paternal chromosomes in the fertilized oocyte can produce viable animals exhibiting a non-Mendelian inheritance pattern. Overexpression in embryos of a single protein regulating chromosome segregation (GPR-1) provides a germline derived clonally from a single parental gamete. We present a collection of strains and cytological assays to consistently generate and track non-Mendelian inheritance. These tools allow reproducible and high-frequency (>80%) production of non-Mendelian inheritance, the facile and simultaneous homozygosis for all nuclear chromosomes in a single generation, the precise exchange of nuclear and mitochondrial genomes between strains, and the assessments of non-canonical mitosis events.

El Mouridi, Sonia et al. (2021) MicroPubl Biol

AlHarbi, Sarah, El Mouridi, Sonia, Frkjr-Jensen, Christian

[MicroPubl Biol, 2021]

For El Mouridi, S; AlHarbi, S; Frkjr-Jensen, C (2021). A histamine-gated channel is an efficient negative selection marker for C. elegans transgenesis. microPublication Biology. 10.17912/micropub.biology.000349.

Wheeler, Bayly et al. (2013) International Worm Meeting "Linking dosage compensation complex assembly to X chromosome gene regulation."

Jorgensen, Erik, Frokjaer-Jensen, Christian, Meyer, Barbara J., Wheeler, Bayly

[International Worm Meeting, 2013]

Dosage compensation (DC) is an essential process required to balance levels of gene expression between the two X chromosomes of females and the single X of males. As DC controls gene expression across the entire X, it serves as an exemplary system to understand mechanisms of long-range gene regulation. In C. elegans, DC is achieved by reducing gene expression from both hermaphrodite X chromosomes by half. DC is enacted by the DC complex (DCC), a condensin complex that is recruited to both hermaphrodite X chromosomes through DNA recruitment elements called rex sites. How these rex sites recruit the DCC, facilitate spreading to neighboring territories, and repress gene expression remains unknown. To understand the relationship between DCC binding and function, we used RNA-seq to identify dosage-compensated genes with high confidence and resolution. Consistent with previous analyses, we find that DCC-regulated genes are interspersed with genes that are not regulated by the DCC. DCC binding within a gene is not predictive of whether that gene will be dosage compensated. Since the DCC acts at a distance to control gene expression, we sought to identify the factors that confer DC status. We have monitored the expression of single-copy gfp transgenes integrated onto autosomes and onto X at different distances from endogenous and engineered rex sites in wild-type and DCC-defective worms. We found that the DCC represses transgene expression at six ectopic sites on X but not on autosomes. Thus, DC controls the expression of introduced and endogenous genes. Furthermore, we show that promoter sequence is not sufficient to determine DC status. We are conducting experiments to address the role of local regulatory elements and chromosome domain structure, as determined by chromosome conformation capture, in dictating DC status. This work will illuminate how regulatory elements on the C. elegans X chromosome achieve appropriate patterns of gene expression chromosome-wide.

Wheeler, Bayly et al. (2015) International Worm Meeting "Regulation of nuclear organization and long-range gene expression by condensin."

Meyer, Barbara J., Frokjaer-Jensen, Christian, Bian, Qian, Anderson, Erika, Jorgensen, Erik, Wheeler, Bayly

[International Worm Meeting, 2015]

The relationship between chromosome structure, nuclear positioning, and long-range gene regulation is poorly understood. To explore this relationship, we dissected X-chromosome-wide gene regulation enacted by a dosage compensation complex (DCC), which represses X transcription in hermaphrodites to balance gene expression between sexes. We inserted transgenes throughout the genome and queried their expression to determine whether different transcriptional environments exist on X and autosomes. Transgenes integrated on X were dosage compensated regardless of position, meaning their expression was equal in wild-type males and hermaphrodites but elevated in dosage-compensation-defective hermaphrodites. This result indicates the X chromosome is broadly permissive for repression, and endogenous genes that escape have special features enabling them to overcome this repression. In contrast, we found no chromosome-wide mechanism to balance X expression with that of autosomes, given that transgenes on X were expressed at half the level of transgenes on autosomes. Repression of X transgenes was independent of their proximity to DCC recruitment sites (rex), highlighting the long-range mechanism of regulation employed by the DCC. We already showed that changes in higher order X-chromosome structure accompany repression of X-linked genes, so we next explored whether spatial positioning of X influences dosage compensation. We first addressed a model of others that rex sites target X to the nuclear periphery in males to increase gene expression, and DCC binding to rex sites in hermaphrodites helps relocate X to the interior, thereby repressing X. Using FISH, we found for both sexes that neither endogenous rex sites on X nor ectopically inserted rex sites on autosomes were preferentially located at the nuclear periphery. Furthermore, though rex insertions on autosomes recruit the DCC, the expression of adjacent genes was not elevated in DCC-depleted animals. These observations disfavor the proposed model. Instead, we found that pairs of distant rex sites interact in a DCC-dependent manner, and interacting rex sites are preferentially located at the nuclear periphery compared to non-interacting sites. Interacting rex pairs associate with nuclear pores, not the lamina. We propose the nuclear pore might act as a scaffold to promote rex site interactions, which in turn influence gene expression by remodeling higher order chromosome structure.