C. elegans Knock-in Services

C. elegans Knock-in Services

The nematode Caenorhabditis elegans is widely used in genetic and biomedical research. DNA fragment insertions can be introduced into the C. elegans genome. Our C. elegans knock-in services include point mutation, floxed allele, degron tagging, fluorescent tagging and immunotagging.

In the past 5 years, InVivo Biosystems has made over 600 fluorescent transgenic lines includuing 15 different fluorophores, 8 of which are variants of GFP.

Fluorescent Tagging. IIntestinal nuclei from late L4 worms in an lmn-1::GFP transgenic strain. The image left is lmn-1 tagged endogenously with GFP. Image courtesy of Dr. André Catic, Baylor College of Medicine.

C. elegans Knock-in Service Offerings

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Fluorescent Tag

Visualize your protein via addition of a fluorescent protein tag at the native locus.

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Immunotag

Use your protein for biochemical studies by adding an immunotag at the native locus.

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Degron Tag

Tag your protein of interest for controlled degradation.

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Point Mutation

The best gene editing method for creating small, precise edits to introduce a small number of nucleotide changes at a target site.

Whole gene humanization

Whole Gene Humanization

Used  to replace a C. elegans gene with an orthologous gene from human or any other organism

C. elegans Knock-in Service Pricing

Build TypeFull Build Candidate Lines Custom Injection Mix
Transgenic Design
Injections
Candidate Screening
Strain Confirmation
Pricing$4,075 and up$2,550 and up$995 and up
Timeline6 weeks and up4 weeks and up3 weeks and up
Note: Additional charge applies for cargo size above 200bp for all packages.

C. elegans Knock-in Service Details

C. elegans Point Mutation Service

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Our Point Mutation service uses CRISPR/Cas9, which is the best gene editing method for creating small, precise edits to introduce a small number of nucleotide changes at a target site.

With this service you can:

  • study a disease-causing mutation
  • humanize a critical amino acid
  • explore the binding site of an enzyme
  • introduce phosphomimetics
  • mutate isoform start sites or make any specific mutation of interest.
Our CRISPR/Cas9 Editing Is Typically Very Efficient

Among the F1 candidates with the Co-CRISPR edit, our average percentage of animals with the target edit was 65.7%.

Edit percentages as high as 95% were observed.

We Can Generate Point Mutations Quickly

For these projects, our average time from injection to sequence confirmation of two independent homozygous lines was 15.3 days.

Our quickest project only took 10 days!

96 Point Mutations In 1 Gene

All 96 mutations were created in the STXBP1 gene (which is associated with epilepsy in humans) via CRISPR.

The worm homolog of STXBP1, unc-18, causes uncoordination and near-complete lack of pharyngeal pumping when knocked out.

The functionality is restored by replacing the worm gene with the coding sequence for human STXBP1.

C. elegans Fluorescent Tagging Service

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Fluorescent tagging of genes is widely used in many model organisms to study which tissues a gene of interest is expressed in, where in the cell a gene is expressed, or to determine whether multiple genes of interest are expressed in the same location. C. elegans is particularly well suited to fluorescence studies because they are transparent. Fluorescence can be easily observed in both live and fixed images.

Knock-in a fluorescent protein at the endogenous locus using CRISPR gene editing technology.

Adding a fluorescent protein tag at the endogenous locus enables you to:

  • detect your protein in-vivo at native gene expression levels
  • ensure that you have the correct expression pattern
  • identify protein-protein interactions utilizing splitGFP
  • confirm degradation of your protein when paired with a degron sequence
  • visualize protein synthesis rates
  • quantify protein levels
Choose From a Variety of Proteins

You can choose from a variety of proteins including YFP, BFP, eGFP, mCherry, mOrange, mScarlet, and many more. All of our fluorescent proteins are optimized for expression in C. elegans via codon optimization and insertion of introns.

Use our fluorescent protein tags in combination with the Fluorescent Protein RNAi to reduce or eliminate protein expression.

Concerned that the addition of a fluorescent protein will inhibit protein function? We can use a 2A or SL2 sequence to separate the 2 components!

Things to consider when ordering your strain:

  • Fluorescent protein best suited for your experiments
  • Best terminus to tag

We are not limited to a fixed list of fluors. If you have a fluor that you would like to use, you can provide us the sequence and we will incorporate it into the design of your transgenic project. If you need your fluorescent transgenics lines to be imaged, we can readily help you with that too! If you are unsure, our genetic engineering experts will be glad to advise you. Contact us.

For immobilization of live animals to image yourself, learn more about NemaGel.

C. elegans Immunotag Service

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The addition of an immunotag at the endogenous locus enables you to quantify your protein without altering the level of gene expression.

Immunotags are added using CRISPR/Cas9 gene editing technology.

You can choose from a variety of tags including FLAG, HA, HIS, TAP, or S-peptide.

All tags have been optimized for expression in C. elegans.

C. elegans Degron Tagging Service

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You can knock-in a degron tag on an endogenous protein or fluorescent protein with our degron tagging service.

Adding a degron tag enables you to:

  • use protein degradation as an complementary method to genetic knockouts or RNAi
  • tag your protein of interested for degradation at the protein level
  • control the timing of protein degradation with an inducer (e.g. auxin, blue light)
  • confirm degradation of your protein when paired with a fluorescent sequence
  • visualize protein localization to organelles or other membranes
Choose From a Variety of Systems

You can choose from a variety of degron inducible and developmental systems including Auxin Inducible Degradation, Photosensitive or blue-light inducible degradation, degron-tagged reporters of membrane topology, and OMA-1 tags that work to degrade protein from the 1-cell stage.

Use our degron tags in combination with the fluorescent tagging to visualize protein expression and degradation.

Things to consider when ordering your strain:

  • Degron tag best suited for your experiments
  • Best terminus to tag

For auxin-inducible degradation, TIR1 is necessary to generate a functional recognition complex and achieve successful target degradation. If you do not have a TIR1-expressing strain ready, we can suggest the most suitable commercially available strain, or we can generate a custom TIR1 strain for you.

If you are unsure, our genetic engineering experts will be glad to advise you. Contact us.

C. elegans Whole Gene Humanization

Whole gene humanization

Whole gene humanization allows you to replace a C. elegans gene with an orthologous gene from human or any other organism. If the human gene rescues the function of the gene deleted-KO, you know there is conserved biology. This is often the first step in a project to create a model to study clinical variants.

We take care to design our humanization projects to preserve endogenous transcription signals so the expression patterns and levels will be maintained. In addition, we have a proprietary sequence optimization protocol that we use to create a transcript that will be expressed well in C. elegans.

Knock-in your human (or other animal) gene of interest at the locus of an orthologous gene using CRISPR gene editing technology.

Creating a whole gene humanized line enables you to:

  • Determine if the human protein is functionally equivalent to the C. elegans protein.
  • Look at drug effects on the human protein in an animal model. 
  • Create a model for the study of clinical variants.

C. elegans  Whole Gene Humanization Service Pricing

Service PackagePriceEst. Delivery Time
Full Build $5,105 and up8 - 12 Weeks
Candidate Lines $4,024 and up6 - 8 Weeks
Custom Injection Mix $2,295 and up
4 - 6 Weeks
Note: Additional charge applies for proteins over 2000 amino acids in size for all packages.

Publications

Point Mutation

  • Single Amino Acid Changes in the Ryanodine Receptor in the Human Population Have Effects In Vivo on Caenorhabditis elegans Neuro-Muscular Function

    Brittany Graham, Marie-Anne Shaw and Ian A. Hope. Front. Genet., 26 February 2020

  • Repurposing the aldose reductase inhibitor and diabetic neuropathy drug epalrestat for the congenital disorder of glycosylation PMM2-CDG

    Sangeetha Iyer, Feba S. Sam, Nina DiPrimio, Graeme Preston, Jan Verheijen, Kausalya Murthy, Zachary Parton, Hillary Tsang, Jessica Lao, Eva Morava2 and Ethan O. Perlstein. Disease Models & Mechanisms (2019) 12.

  • Repurposing the aldose reductase inhibitor and diabetic neuropathy drug epalrestat for the congenital disorder of glycosylation PMM2-CDG

    Iyer S, Murthy K, Parton Z, Tsang H, Sam FS. bioRxiv. 2019 May 3; 1-29

  • Regulation of the sperm-to-oocyte transition in Caenorhabditis briggsae hermaphrodites by the Cbr-met-2 and Cbr-fem-3 genes

    Berenson AL, Baird SE. Mol Reprod Dev. 2018 Jun;85(6):532-542.

  • NALCN channelopathies: Distinguishing gain-of-function and loss-of-function mutations

    Bend, EG; Si, Y; Stevenson, DA; Bayrak-Toydemir, P; Newcomb, TM; Jorgensen, EM; Swoboda, KJ. Neurology. 2016 Sep 13;87(11):1131-9.

  • An RNAi-based suppressor screen identifies interactors of the Myt1 ortholog of Caenorhabditis elegans

    Allen, AK; Nesmith, JE; Golden, A. G3 (Bethesda). 2014 Oct 8;4(12):2329-43.

  • LEM-3 is a midbody-tethered DNA nuclease that resolves chromatin bridges during cytokinesis

    HONG, Y; Sonneville, R; Wang, B; Scheidt, V; Meier, B; Woglar, A; Demetriou, S; Labib, K; Jantsch, V; Gartner, A. Nat Commun. 2018 Feb 20;9(1):728.

Tagging

  • Diet-responsive Transcriptional Regulation of Insulin in a Single Neuron Controls Systemic Metabolism

    Ava Handley, Qiuli Wu, Tessa Sherry, Roger Pocock. bioRxiv. August 31, 2019.

  • The myosin light-chain kinase MLCK-1 relocalizes during Caenorhabditis elegans ovulation to promote actomyosin bundle assembly and drive contraction

    Charlotte A. Kelley, Alison C. E. Wirshing, Ronen Zaidel-Bar, and Erin J. Cram. MBoC. August 08, 2018: 1917-2035

  • The exocyst complex and Rab5 are required for abscission by localizing ESCRT III subunits to the cytokinetic bridge

    Harsh Kumar, Kumari Pushpa, Amrita Kumari, Kuldeep Verma, Rajaiah Pergu, Sivaram V. S. Mylavarapu. J. of Cell Science. 17 July 2019

  • Syncytial germline architecture is actively maintained by contraction of an internal actomyosin corset

    Priti A, Ong HT, Toyama Y, Padmanabhan A, Dasgupta S, Krajnc M, Zaidel-Bar R. Nat Commun. 2018 Nov 8;9(1):4694.

  • Disruption of the Caenorhabditis elegans Integrator complex triggers a non-conventional transcriptional mechanism beyond snRNA genes

    Gómez-Orte E, Sáenz-Narciso B, Zheleva A, Ezcurra B, de Toro M, López R, Gastaca I, Nilsen H, Sacristán MP, Schnabel R, Cabello J. PLoS Genetics. 2019 Feb 26;15(2):e1007981.

  • The conserved LEM-3/Ankle1 nuclease is involved in the combinatorial regulation of meiotic recombination repair and chromosome segregation in Caenorhabditis elegans

    Hong Y, Velkova M, Silva N, Jagut M, Scheidt V, Labib K, Jantsch V, Gartner A. PLoS Genetics. 2018 June 7;14(6):e1007453.

  • The exocyst complex and Rab5 are required for abscission by localizing ESCRT III subunits to the cytokinetic bridge

    Kumar H, Pushpa K, Kumari A, Verma K, Pergu R, Mylavarapu SVS. J. Cell Science. 2019 July 17;132(14).

  • LEM-3 is a midbody-tethered DNA nuclease that resolves chromatin bridges during late mitosis

    Hong Y, Sonneville R, Wang B, Scheidt V, Meier B, Woglar A, Demetriou S, Labib K, Jantsch V, Gartner A. Nat Commun. 2018 Feb 20;9(1):728.

  • The myosin light chain kinase, MLCK-1 relocalizes during C. elegans ovulation to promote actomyosin bundle assembly and drive contraction

    Kelley CA, Wirshing ACE, Zaidel-Bar R, Cram EJ. Mol Biol Cell. 2018 Aug 8;29(16):1975-1991.

  • CUL-2LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis

    Sonneville, R; Moreno, SP; Knebel, A; Johnson, C; Hastie, CJ; Gartner, A; Gambus, A; Labib, K. Nat Cell Biol. 2017 May;19(5):468-479.

  • Olfactory stem cells reveal MOCOS as a new player in autism spectrum disorders

    Féron, F; Gepner, B; Lacassagne, E; Stephan, D; Mesnage, B; Blanchard, MP; Boulanger, N; Tardif, C; Devèze, A; Rousseau, S; Suzuki, K; Izpisua Belmonte, JC; Khrestchatisky, M; Nivet, E; Erard-Garcia, M. Mol Psychiatry. 2016 Sep;21(9):1215-24.

  • Ribonuclease-Mediated Control of Body Fat

    Habacher, C; Guo, Y; Venz, R; Kumari, P; Neagu, A; Gaidatzis, D; Harvald, EB; Færgeman, NJ; Gut, H; Ciosk, R. Dev Cell. 2016 Nov 7;39(3):359-369.

  • Replication-Independent Histone Variant H3.3 Controls Animal Lifespan through the Regulation of Pro-longevity Transcriptional Programs

    Piazzesi, A; Papić, D; Bertan, F; Salomoni, P; Nicotera, P; Bano, D. Cell Rep. 2016 Oct 18;17(4):987-996.

  • The C. elegans VAPB homolog VPR-1 is a permissive signal for gonad development

    Cottee, PA; Cole, T; Schultz, J; Hoang, HD; Vibbert, J; Han, SM; Miller, MA. Development. 2017 Jun 15;144(12):2187-2199.

  • Mechanism of biofilm-mediated stress resistance and lifespan extension in C. elegans

    Smolentseva, O; Gusarov, I; Gautier, L; Shamovsky, I; DeFrancesco, AS; Losick, R; Nudler, E. Sci Rep. 2017 Aug 2;7(1):7137.

  • Regulation of DJ-1 by Glutaredoxin 1 in Vivo: Implications for Parkinson’s Disease

    Johnson, WM; Golczak, M; Choe, K; Curran, PL; Miller, OG; Yao, C; Wang, W; Lin, J; Milkovic, NM; Ray, A; Ravindranath, V; Zhu, X; Wilson, MA; Wilson-Delfosse, AL; Chen, SG; Mieyal, JJ. Biochemistry. 2016 Aug 16;55(32):4519-32.

  • Caenorhabditis elegans BUB-3 and SAN-1/MAD3 Spindle Assembly Checkpoint Components Are Required for Genome Stability in Response to Treatment with Ionizing Radiation

    Bertolini, S;Wang, B;Meier, B;Hong, Y;Gartner, A. G3 (Bethesda). 2017 Dec 4;7(12):3875-3885.

  • A Bystander Mechanism Explains the Specific Phenotype of a Broadly Expressed Misfolded Protein

    Klabonski, L;Zha, J;Senthilkumar, L;Gidalevitz, T. PLoS Genet. 2016 Dec 7;12(12):e1006450.

  • A Differentiation Transcription Factor Establishes Muscle-Specific Proteostasis in Caenorhabditis elegans

    Bar-Lavan, Y; Shemesh, N; Dror, S; Ofir, R; Yeger-Lotem, E; Ben-Zvi, A. PLoS Genet. 2016 Dec 30;12(12):e1006531.

  • The nphp-2 and arl-13 genetic modules interact to regulate ciliogenesis and ciliary microtubule patterning in C. elegans

    Warburton-Pitt, SR; Silva, M; Nguyen, KC; Hall, DH; Barr, MM. PLoS Genet. 2014 Dec 11;10(12):e1004866.

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