Semple, Jennifer I. et al. (2011) International Worm Meeting "Update on the use of drug selection in nematodes."

Lehner, Ben, Semple, Jennifer I.

[International Worm Meeting, 2011]

We recently described the use of puromycin to select large populations of transgenic worms from lines carrying extrachromosomal arrays with the resistance gene (Nat Methods. 2010; 7(9):725-7.). Now, we have extended the technique to select for transgenic worms following bombardment. We have introduced some modifications in the standard bombardment protocol and created a three way Gateway compatible plasmid expressing both puromycin and neomycin resistance genes to make drug selection possible, despite the extremely high dilution ratio of transgenic to non-transgenic worms. The new protocol makes it possible to pick adult F1 generation worms only 8 days after bombardment, significantly faster than the most widely used selection protocol with unc-119. Furthermore, this drug selection regime does not require any particular genetic background and we have successfully generated lines with integrated transgenes in worms from various different strains and species, namely C. elegans (N2, CB4856), C. briggsae (AF16, HK104) and C. remanei (PB4641).

Semple, Jennifer Isabel et al. (2009) International Worm Meeting "A simple drug selection system for C. elegans."

Garcia-Verdugo, Rosa, Lehner, Ben, Semple, Jennifer Isabel

[International Worm Meeting, 2009]

The commonly used transgenesis selection markers in worms involve the introduction, or rescue, of mutant phenotypes. Such mutations make the growth of the worms more labor intensive and can possibly interfere with the analysis of the transgene of interest. In addition, rescue of a mutant phenotype, such as the widely used unc-119 system, is limited to species that contain an appropriate mutant worm strain. Here we report the development of a drug selection system in order to address these issues. Our experiments were carried out with mosaic animals expressing the gene from an extra-chromosomal array, suggesting that this system would be extremely useful for large scale genomic or proteomic studies where one could combine the ease of generating worms carrying extra-chromosomal transgenes with an easy protocol for enriching large populations of transgenic worms. We also anticipate that this selection system will be useful for other applications, such as stable worm transformation by bombardment, which requires selection of rare transformation events from among a large number of worms.

Jennifer I Semple et al. (2009) European Worm Neurobiology Meeting "A simple drug selection system for C. elegans"

Jennifer I Semple, Ben Lehner, Rosa Garcia-Verdugo

[European Worm Neurobiology Meeting, 2009]

Available methods for selecting transgenic C. elegans strains are based on the rescue of a detrimental mutation such as unc-119 or the use of visible markers such as GFP. These approaches are often labour intensive and cannot be easily applied to other species. We have developed a selection system using the antibiotic puromycin. The selection is achieved with a plasmid that expresses the antibiotic resistance gene under the control of a ribosomal gene promoter. When different worm strains carrying an extrachromosomal array containing the puromycin resistance gene are treated with puromycin in liquid medium for 4 days, we routinely obtain populations in which 80-100% of the worms carry the transgene with a recovery rate of >50% of transgenic worms from the original population. We show that transgene selection with our vector can be carried out in at least two species of worms: C. elegans and C. briggsae. In summary, we describe a positive selection system based on puromycin which: - Allows for the easy and rapid selection of rare transformation events - Is independent of genetic background - Can be applied to other Caenorhabditis species - Constitutes a way to enrich large populations of worms carrying non-integrated transgenes.

Jennifer Isabel Semple et al. (2010) Evolutionary Biology of Caenorhabditis and Other Nematodes "RAPID TRANSGENE SELECTION WITH PUROMYCIN IN CAENORHABDITIS SPECIES"

Ben Lehner, Rosa Garcia-Verdugo, Jennifer Isabel Semple

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2010]

We have developed a drug selection system for transgene expression in nematodes. Worms carrying an extrachromosomal array containing the puromycin resistance gene are efficiently selected from a mixed population. This approach has a number of advantages over existing methods for transgene selection: 1) Large numbers of transgenic worms can be easily selected without the need for expensive, specialised equipment such as a worm-sorter. 2) Strains carrying extrachromosomal arrays with puromycin resistance can be maintained on puromycin plates without needing to pick individual worms. 3) Puromycin selection does not require a particular genetic background, eliminating the need to generate particular mutant strains for different species, which are often laborious to work with. We have successfully tested puromycin selection in both C. elegans and C. briggsae and anticipate that this system could be useful for transgenesis in a variety of Caenorhabditis species. 4) We have developed a protocol for rapid drug selection following bombardment of N2 worms.

Watts JL et al. (1994) Worm Breeder's Gazette "zu170 Defines a New Gene, par-6, and Can Act as a Suppressor of par-2"

Watts JL, Kemphues KJ

[Worm Breeder's Gazette, 1994]

zul70 defines a new gene, par-6, and can act as a suppressor of par-2 Jennifer Watts and Ken Kemphues Genetics and Development, Cornell University, Ithaca NY 14853

Lehner, Ben et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Rapid selection and maintenance of transgenic Caenorhabditis species using drug selection"

Garcia-Verdugo, Rosa, Lehner, Ben, Semple, Jennifer

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

We have developed a drug selection system for transgene expression in nematodes. Worms carrying an extrachromosomal array containing the puromycin resistance gene are efficiently selected from a mixed population. This approach has a number of advantages over existing methods for transgene selection: 1) Large numbers of transgenic worms can be easily selected without the need for expensive, specialised equipment such as a worm-sorter. 2) Strains carrying extrachromosomal arrays with puromycin resistance can be maintained on puromycin plates without needing to pick individual worms. 3) Puromycin selection does not require a particular genetic background, eliminating the need to generate particular mutant strains for different species, which are often laborious to work with. We have successfully test ed puromycin selection in both C. elegans and C. briggsae and anticipate that this system could be useful for transgenesis in a variety of Caenorhabditis species. 4) We have developed a protocol for rapid drug selection following bombardment of N2 worms.

Isiaka, B.Nafisat. et al. (2019) International Worm Meeting "Examining transcription dynamics at the single molecule level in individual promoters."

Semple, Jennifer, Meister, Peter, Krebs, Arnaud, Isiaka, B.Nafisat.

[International Worm Meeting, 2019]

Transcription initiation is a dynamic, multi-step process, requiring the ordered recruitment of multiple factors to the core promoter. The highly dynamic nature of transcription means that at any given moment, a particular promoter might be in one of multiple states in different cells, resulting in heterogeneous signals from a population of cells. Most techniques that study transcription initiation, such as chromatin-IP or GRO-seq provide population average measurements of the transcriptional process. To really understand transcription regulation, it is necessary to examine changes in promoter state at the single molecule level. To achieve this, we applied to C. elegans a technique known as "dual enzyme Single Molecule Footprinting (dSMF)". The method involves in vitro methylation of chromatin in intact nuclei with bacterial CpG and GpC methyltransferases. Closed chromatin bound by proteins is protected from methylation, whereas accessible DNA is methylated. After bisulfite conversion, specific amplicons, or genome-wide DNA libraries are sequenced to identify footprints of accessible DNA. Correlation of dSMF data with publicly available genome wide datasets shows that dSMF footprints coincide with nucleosome free regions in promoters and other genomic regions such as HOT sites. To identify what the different footprints represent in terms of promoter states in the transcription cycle, we then inhibit individual steps of the transcription cycle either with chemicals or by blocking engineered cyclin-dependent kinases with ATP analogs. Upon mapping the promoter occupancy states in transcription inhibited conditions, we are able to correlate the different promoter footprints with specific protein complexes present during the different steps of transcription initiation. Our data strengthen our understanding of how chromatin and gene structure regulate gene expression at the level of transcription initiation.

Isiaka, Bolaji et al. (2021) International Worm Meeting "In vivo DNA Topology and Transcriptional Regulation in Caenorhabditis elegans"

Isiaka, Bolaji, Gilbert, Nick, Meister, Peter, Semple, Jennifer

[International Worm Meeting, 2021]

DNA is an antiparallel double helix; its transcription requires separating the two strands. This creates supercoils, underwound and overwound duplex DNA, which accumulate behind and in front of the transcription bubble, respectively. Overwound supercoils are detrimental to helix opening and transcription bubble formation, as they increase the energy necessary to separate strands. In vivo, these structures are resolved by the action of topoisomerases which release supercoils from DNA. Conversely, the action of condensins, molecular machines creating loops in chromatin, has been shown in vitro to increase supercoiling. The interplay between supercoiling, transcription and condensin action could be a powerful regulator of gene expression. To understand this interplay, we mapped supercoiling genome-wide in vivo in L3 larvae using biotinylated 4,5,8-trimethylpsoralen (bTMP). bTMP preferentially intercalates in negatively supercoiled DNA and is crosslinked to it using UV illumination. Using biotin pulldown, we can then enrich for negatively supercoiled DNA before high-throughput sequencing. Our results show highly reproducible supercoiling profiles. At chromosome scale, we did not observe a significant difference in supercoiling between chromosomes. In contrast, telomeric thirds of autosomes (perinuclear heterochromatic B domains) show a lower bTMP enrichment than at central domains (euchromatic A domains), in agreement with higher transcription levels in euchromatin. At the gene level, bTMP enrichment shows two peaks, one 5' of the transcription start site (TSS) and one at the transcription end site (TES). This suggests the accumulation of negative supercoils in the promoter region of genes and at the end of the transcription units. In agreement with a transcriptional cause, the bTMP enrichment profile along the genes correlates with gene expression levels. In hermaphrodite animals, a variant of condensin I called condensin IDC, is part of the dosage compensation complex which downregulates gene expression of X-linked genes. Condensins I/IDC purified from nematodes can induce the formation of supercoils in vitro. In vivo, we observe differential bTMP enrichment at X-linked TSS, coinciding with the location of the condensin IDC. This suggests that supercoiling may link the X-specific transcriptional repression and condensin loading.

Isiaka, Bolaji et al. (2019) International Worm Meeting "Examining transcription dynamics at the single molecule level in individual promoters."

Isiaka, Bolaji, Meister, Peter, Krebs, Arnaud, Semple, Jennifer

[International Worm Meeting, 2019]

Transcription initiation is a dynamic, multi-step process, requiring the ordered recruitment of multiple factors to the core promoter. The highly dynamic nature of transcription means that at any given moment, a particular promoter might be in one of multiple states in different cells, resulting in heterogeneous signals from a population of cells. Most techniques that study transcription initiation, such as chromatin-IP or GRO-seq provide population average measurements of the transcriptional process. To really understand transcription regulation, it is necessary to examine changes in promoter state at the single molecule level. To achieve this, we applied to C. elegans a technique known as "dual enzyme Single Molecule Footprinting (dSMF)". The method involves in vitromethylation of chromatin in intact nuclei with bacterial CpG and GpC methyltransferases. Closed chromatin bound by proteins is protected from methylation, whereas accessible DNA is methylated. After bisulfite conversion, specific amplicons, or genome-wide DNA libraries are sequenced to identify footprints of accessible DNA. Correlation of dSMF data with publicly available genome wide datasets shows that dSMF footprints coincide with nucleosome free regions in promoters and other genomic regions such as HOT sites. To identify what the different footprints represent in terms of promoter states in the transcription cycle, we then inhibit individual steps of the transcription cycle either with chemicals or by blocking engineered cyclin-dependent kinases with ATP analogs. Upon mapping the promoter occupancy states in transcription inhibited conditions, we are able to correlate the different promoter footprints with specific protein complexes present during the different steps of transcription initiation. Our data strengthen our understanding of how chromatin and gene structure regulate gene expression at the level of transcription initiation.

Giordano-Santini, Rosina et al. (2010) Worm Breeder's Gazette "Antibiotic markers for rapid selection and easy maintenance of transgenic nematodes"

Lehner, Ben, DUPUY, Denis, Semple, Jennifer, Giordano-Santini, Rosina

[Worm Breeder's Gazette, 2010]