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Robust expression of transgenes in the C. elegans germline through a simple microinjection protocol.

Luisa Cochella2,4, Baris Tursun3,4, Nikolaos Stefanakis1,4, Dylan Rahe1,4, Inés Carrera1,4 and Oliver Hobert1
1Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York NY, 2IMP – Research Institute of Molecular Pathology, Vienna, Austria,3Berlin Institute for Medical Systems Biology (BIMSB) at Max Delbrück Center (MDC), Berlin, Germany, 4These authors contributed equally to this work

Correspondence to: or38@columbia.edu (or38@columbia.edu)

The ease of generating transgenic animals is one of the key experimental advantages of C. elegans as a model organism. Typically, microinjection of DNA encoding the desired transgene results in heritable extra-chromosomal arrays that provide robust expression in the somatic tissues of C. elegans. Achieving expression in the germline with such arrays is however, less successful due to silencing of repetitive sequences in this tissue. In order to “dilute” the repetitiveness of the array to achieve germline expression, addition of N2 genomic or foreign DNA in the array has been shown to be effective (Kelly et al. (1995) WBG 14(1): 64; Kelly et al., 1997; Lam et al. 2006). Another way to achieve germline expression is the production of low copy-number arrays using methods such as the generation of single-copy arrays by microparticle bombardment or transposon-mediated chromosomal integration approaches (Praitis et al., 2001; Frokjaer-Jensen et al., 2008). However, in contrast to microinjection protocols, these approaches often produce low expression levels of reporters such as fluorescent proteins and they also require the use of specific genetic backgrounds.

In our lab we have been able to obtain stable germline expression from several extrachromosomal and integrated transgenic reporters using a microinjection-based protocol, in which we include either sonicated or PvuII digested E. coli genomic DNA as carrier DNA in the injection mix.

We obtain E. coli genomic DNA using standard methods or commonly used genomic DNA kits. We sonicate the DNA to obtain a smear of DNA sizes centered at around 1kb. Fragmentation of E. coli genomic DNA can be also performed by complete PvuII digestion. Both ways give stable lines with germline expression in our hands. We then pass the fragmented genomic DNA through DNA purification columns. The final concentration of bacterial DNA in the injection mix is from 100-150 ng/ul and reporters and co-injection markers are linearized and injected at the concentrations shown in Table 1. We have successfully obtained germline expression of small reporters (plasmid based) as well as fosmid reporters. We also found no effect on germline expression using either DNA methylation competent or deficient E. coli strains as genomic DNA source.

In Table 1, we show transgenes injected using our protocol that gave stable germline expression, some of which had previously been characterized with respect to copy number using aCGH (Cochella et al. (2011) WBG 18(4)). In this table there are transgenes that have low copy number, like tbb-1 (otEx4548) (mean copy number 5/array), and high copy number, like hs::che-1 (otIs284) (mean copy number 11/chromosome). This suggests that for the protocol that we follow, copy numbers within the range that we have obtained can support germline expression.

Although the addition of bacterial DNA to arrays is not novel, as far as we have followed these strains, we do not observe silencing of reporters after several generations as noted before (Kelly et al. (1995) WBG 14(1): 64). In addition, using bacterial DNA instead of N2 genomic has also the advantage of not introducing wild-type worm genes into mutant backgrounds.

We cannot exclude that expression of some genes may be more sensitive to copy number and/or genomic context and therefore may still require low/single-copy insertions to produce expression in the germline. However, we believe that this is a robust and simple enough approach to be considered as the first thing to try when germline expression is expected/required. Therefore, we hope that our method will help C. elegans researchers to obtain germline expression of transgenes using microinjection techniques.

Figures



Figure 1: Table 1: List of reporters that give germline expression using our microinjection protocol.

References

Frokjaer-Jensen C, Davis MW, Hopkins CE, Newman BJ, Thummel JM, Olesen SP, Grunnet M, Jorgensen EM. (2008) Single-copy insertion of transgenes in Caenorhabditis elegans. Nature Genet 40, 1375-1383. PubMed

Kelly WG, Xu S, Montgomery MK, Fire A. (1997) Distinct requirements for somatic and germline expression of a generally expressed Caernorhabditis elegans gene. Genetics 146, 227-238. PubMed

Lam N, Chesney MA, Kimble J. (2006) Wnt signaling and CEH-22/tinman/Nkx2.5 specify a stem cell niche in C. elegans. Curr Biol 16, 287-295. PubMed

Praitis V, Casey E, Collar D, Austin J. (2001) Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157, 1217-1226. PubMed

Tursun B, Patel T, Kratsios P, Hobert O. (2011) Direct conversion of C. elegans germ cells into specific neuron types. Science 331, 304-308. PubMed

How to measure lethargus quiescence without monopolizing the dissection microscopes in your lab

Huiyan Huang1, Chen-Tseh Zhu2 and Anne Hart1
1Department of Neuroscience, Brown University, Providence RI, 2Department of Ecology and Evolutionary Biology, Brown University, Providence RI

Correspondence to: Anne Hart (anne_hart@brown.edu)

During C. elegans lethargus, a sleep-like behavior called quiescence occurs in short bouts during which animals spontaneously pause locomotion and feeding (Raizen et al., 2008). Multiple laboratories are interested in studying quiescence, however monitoring this behavior is problematic as high-magnification images must be captured for individual animals over a long period of time. Using a dissection scope with a camera to measure quiescence is generally expensive and monopolizes microscopes that would normally be used for other purposes. We describe here a set-up that can be used to follow individual animals without using a dissection microscope. We load up to 6 late-L4 animals into simple, reusable microfluidic chambers and analyze images captured every 10 seconds throughout lethargus (Singh et al., 2011). MATLAB scripts for acquiring and analyzing the images are available upon request.

 

Equipment (total cost: $2072 without computer)

1. 76mm HH Dual Illuminated Track Stand (Halogen/Halogen) with N-Type Adapter from Howard Electronic Instruments (#HEI-TS-HH-N) for $462. This is a special order that is not available in the catalog. The sales manager is willing to custom-assemble this stand.

2. AVT Guppy Pro F-503 color CCD camera from Edmund Optics (# NT68-572) for $760.

AVT camera driver compatible with MATLAB needs to be acquired from the AVT website.

3. 1/4-20 Mounting Adapter for Guppy Pro from Edmund Optics (#NT68-585) for $45.

4. 50 mm Fixed Focal Length 10 MegaPixel Lens with Manual Focus and Iris, C-mount, 2/3in. format, F-stop: 2.0, Filter: 30.5, Pitch: 0.5 from Graftek Imaging (# 1-19558) for $747.

5. Schneider C-mount Extension Tube Kit from B&H (B&H # SCCMETK, Mfr# 21-06010) for $58. The kit includes 0.5/1/5/10/20/40 mm tubes.

6. Computer and adapters:

a) If you are concerned about budget and have plenty of space, a desktop computer with a Firewire card adapter and operating system that meets MATLAB system requirements will do. The cost for both the desktop and Firewire card adapter starts at $500.

b) A laptop can be used to save space but is more expensive. Purchase a laptop with an operating system that meets MATLAB system requirements and has an Expresscard slot. Also, purchase a SIIG NN-EC2812-S2 2-port Firewire 800 ExpressCard/54 from Newegg (# N82E16839150025) for $78 and Power adapter(# N82E16839150034) for $22.

 

Instructions for assembly:

1. Assemble the track stand:
Assemble the track stand according to the instructions. The top lighting does not need to be assembled since it is not necessary.

2. Assemble the camera and lens:
The minimum focusing distance for the lens is 10 cm and the field-of-view at minimum focusing distance is 14.3 mm × 19.1 mm. Currently, our chip for quiescence chamber is 4 mm × 6 mm. To achieve a shorter focusing distance and smaller field-of-view, connect the camera and lens with a combination of extension tubes (20 mm and 5 mm). You can change extension tubes when working with microfluidic chips of different sizes.

3. Mount the camera to track stand:
Mount the camera to the N-type adapter with the mounting adapter. Make sure the camera and lens are perpendicular to the track stand base.

4. Connect the camera to a computer with a Firewire cable.

5. Install MATLAB with Image Acquisition Toolbox and AVT camera driver compatible with MATLAB.

References

Raizen DM, Zimmerman JE, Maycock MH, Ta UD, You YJ, Sundaram MV, Pack AI. (2008) Lethargus is a Caenorhabditis elegans sleep-like state. Nature 451, 569-472.   PubMed

Singh K, Chao MY, Somers GA, Kamatsu H, Corkins ME, Larkins-Ford J, Tucey T, Dionne HM, Walsh MB, Beaumont EK, Hart DP, Lockery SR, Hart AC. (2011) C. elegans Notch signaling regulates adult chemosensory response and larval molting quiescence. Curr Biol 21, 825-834.  PubMed

C9ORF72/F18A1.6 is required for normal development and locomotion in Caenorhabditis elegans.

Xin Wang1 and Edgar Buttner1,2
1Pharmacogenomics Section, Molecular Pharmacology Laboratories, McLean Hospital and Harvard Medical School, Belmont MA, 2Departments of Neurology and Psychiatry, McLean Hospital and Harvard Medical School, Belmont MA

Correspondence to: Edgar Buttner (ebuttner@partners.org)

Mutations in the human C9ORF72 gene cause frontotemporal dementia and amyotrophic lateral sclerosis (Boeve et al., 2012; Renton et al., 2011). The normal and abnormal functions of this gene are unknown, and data from animal models have not been previously reported. We have characterized developmental and behavioral phenotypes in mutants of F18A1.6, the C. elegans homolog of C9ORF72. The in-frame insertion/deletion allele ok3062 was backcrossed to wild type six times, and no gross abnormalities were noted in development and fertility. In contrast, feeding RNAi of F18A1.6 produced quantifiable developmental delay in an rrf-3 background, compared to empty-vector control. F18A1.6 and F18A1.7 appear to compose an operon, but RNAi of F18A1.7 produced no gross defects in development. RNAi of F18A1.6 did not affect brood size.

RNAi of F18A1.6 impaired locomotion in a thrashing assay, compared to empty-vector control. In contrast, ok3062 animals displayed enhanced thrashing, compared to N2. We are currently quantifying thrashing behavior in animals exposed to RNAi of F18A1.7. Our laboratory is also generating transgenic animals to characterize the expression pattern of F18A1.6 and to test for rescue by human C9ORF72. GFP reporter strains are being screened for neuroanatomical defects that might underlie the thrashing phenotypes of F18A1.6 mutants.

Figures



Figure 1: Figure A, The ok3062 insertion/deletion allele enhances thrashing. EAB112 was generated by backcrossing ok3062 to N2 six times. Animals were soaked in halocarbon oil to completely remove bacteria prior to a 60 sec. adaptation on bacteria-free agar plates with M9. Thrashes were scored for 30 sec. in animals showing continuous movement. Two independent experiments were performed (n=24 for N2 & n=23 for EAB112 at one day post-L4; n=14 for N2 & n=13 for EAB112 at two days post-L4). Figure B, RNAi of F18A1.6 impairs thrashing (n=24 for empty-vector control & n=28 for RNAi of F18A1.6). Figure C, RNAi of F18A1.6 produces developmental delay. Results depicted are from day 3 after eggs were laid. Experiments were performed at 20 degrees.

References

Boeve FB, Boylan BB, Graff-Radford NR, DeJesus-Hernandez M, Knopman DS, Pedraza O, et al. (2012) Characterization of frontotemporal dementia and/or amyotrophic lateral sclerosis associated with the GGGGCC repeat expansion in C9ORF72. Brain 135, 765-783. PubMed

Renton AE, Majounie E, Waite A, Simón-Sánchez J, Rollinson S, Gibbs JR, et al. (2011) A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257–268. PubMed

Snodprot-a protein changing chemotaxis and locomotion of Caenorhabditis elegans

Hanying Yu
1Department of Petroleum Engineering, Northeast Petroleum University, Daqing, China

Correspondence to: Hanying Yu (yu_hanying@yahoo.cn)

Snodprot, which was a member of the cerato-platanin family, was found to change the chemotaxis and increase the body-bend frequency of Caenorhabditis elegans in a concentration-dependent manner in the study on the identification of snodprot gene of nematophagous fungi Dactylellina cionopaga (Yu et al., 2012). In L1 growth assay, stain with SYBR Green and lethality assay, snodprot did not affect the growth of the nematodes and kill the cells or the nematodes. The protein presented to reverse the functions of serotonin, but could not reverse the functions of octopamine, an antagonist of serotonin.

The chemotaxis-changing mechanism of snodprot might involve related neurons, the downstream signal transduction pathway, and regulatory elements because the attraction substance applied in this chemotaxis assay was NaCl. In the water-soluble chemotaxis of C. elegans ASE was the major neuron, and there was a weak residual response distributed over numerous classes of neurons, including ADF, ASG, ASI, ASK, and ASJ ( Bargmann, 2006). The signal transduction of ASE involved tax-4, tax-2, daf-11, and cGMP, the regulators of which are osm-9, gpc-1, tax-6, ttx-4, and adp-1. The receptors and the corresponding G proteins of the salts and water-soluble attractants might have been guanylate cyclases and gpa-3 (Bargmann, 2006).

The neurotransmitter serotonin regulates a wide range of behaviors in C. elegans, including egg laying, male mating, and locomotion (inhibition of movement) besides pharyngeal pumping (Chase and Koelle, 2007). The fact that snodprot could increase the locomotion of the nematodes significantly and in a concentration-dependent manner is consistent with the results of reversing the functions of serotonin of the protein. Expression of F01E11.5 (tyra-2), the receptor for serotonin and octopamine, was identified in pharyngeal neurons MC and NSM, amphid neurons ASE, ASG, ASH, ASI, and other neurons, including PVD, CAN, and ALM (Chase and Koelle, 2007).

The neurons are highly interconnected, and the neural networks for the exploratory and taxis behaviors partially overlap (White et al., 1986; Samuel and Sengupta, 2005). The transition to travelling search can involve the ASI, ASE, ADF, and ASH sensory neurons. The prominent targets of the ASE and AIY interneurons and additional targets, including the AIA and AIB interneurons of C. elegans, all regulate pirouettes (Bargmann, 2006). Samuel and Sengupta have also reported that the major neuronal components of the sensorimotor circuits of C. elegans, which mediate local search and long-range roaming behaviors, include chemosensory neurons ASI, AWC, and ASK, and interneurons AIB and AIY (Samuel and Sengupta, 2005).

It can be concluded that both ASE and ASI neutrons are involved in the process of chemotaxis to NaCl, the response to serotonin, and the locomotion of C. elegans. Thus the mechanism affected by snodprot is to be expected to combine competitively with serotonin in ASE or ASI neurons though the definitive combination site of snodprot awaits the confirmations of the immunity precipitation and other experiments.

References

Bargmann CI. (2006) Chemosensation in C. elegans. WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.123.1, http://54.165.141.74/.  PubMed

Chase DL, Koelle MR. (2007) Biogenic amine neurotransmitters in C. elegans. WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.132.1, http://54.165.141.74/.  PubMed

Samuel ADT, Sengupta P. (2005) Sensorimotor integration: locating locomotion in neural circuits. Curr Biol 15, R341-R343.  PubMed

White JG, Southgate E, Thomson JN, Brenner S. (1986) The structure of the nervous system of the nematode Caenorhabditis elegans. Pilos Tran. R Soc Lond B, 314, 1-340.  PubMed

Yu HY, Duan JX, Wang B, Jiang XZ. (2012) The function of snodprot in the cerato-platanin family from Dactylellina cionopaga in nematophagous fungi. Biosci Biotechnol Biochem 76, 1835-1842.  PubMed

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