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C. elegans Knockout Services

C. elegans Knockout Services

Knockouts are the bread and butter of reverse genetic studies. They allow researchers to study the effects of the absence of a particular gene, protein segment, amino acid, or regulatory element, thereby enabling the researcher to determine their function. Gene knockouts are a commonly used tool for biologists to understand gene function. Examination of phenotypes when the gene is deleted can reveal insights into what role that gene plays in the organism.

Full, Partial and Conditional Gene Deletion

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Full Gene Deletion

For research requiring a true null mutant, we recommend working with a precise KO to ensure the accuracy of your data. Many traditional null mutants rely on an early stop, which may not create a true null. Any genomic region can be targeted for deletion.

We can delete the entire coding sequence of a gene using CRISPR/Cas9. You can then study the phenotype of the true null allele with no concern that any protein function remains. Our transgenic designers and process ensure that each knockout we make is exactly the right strain to answer the research question.

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Partial Gene Deletion

The Precise Deletion service uses CRISPR/Cas9 genome editing to remove a small defined region of DNA. This can be useful for deleting protein domains, DNA regulatory regions, or any other sequence of your choice.

Deletions can also be performed in coding regions for functional analysis of protein domains or to isolate isoform function. Transcription factor binding sites or introns can be deleted to reveal information about gene regulation.

Floxed allele

Conditional Gene Deletion

Floxed alleles can be created for a tissue- or temporal- specific deletion of your gene of interest. This is a powerful tool for studying embryonic lethal genes or for understanding how your gene of interest functions in different tissues. CRISPR/Cas9 is used to insert loxP sites flanking the region to be deleted.

This line can then be crossed with a Cre-line expressing the Cre recombinase under a specific promoter or injected with a plasmid containing Cre recombinase. The Cre recombinase promotes recombination of the two loxP sites and the region between the sites is removed from the genome.

Conditional alleles

  • Used to make spatiotemporal gene deletions
  • Flank gene of interest with LoxP sites
  • Inject a plasmid or cross into strain that expresses Cre recombinase where/when/how you want loss-of-function to occur

We are able to build custom Cre recombinase-expressing lines to compliment your new floxed allele.

A client wanted a knockout (KO) of an embryonic lethal gene. We could not make this line using our standard methods. Instead, we inserted two loxP sites. One in the first intron of the gene and the second in the 3’utr. After we confirmed this line by PCR and sequencing, we injected this line with a ubiquitously expressing Cre Recombinase plasmid.

We found while the uninjected animals could reproduce, the Cre injected animals did not (Figure A). We tested recombination by PCR and found that only the Cre injected progeny showed recombination of the loxP sites (Figure B).

C. elegans Mutation - Floxed Allele FigA
Fig. A
C. elegans Mutation - Floxed Allele FigB
Fig. B

This line could be used to study KO of this gene in adulthood or in specific tissues, something that was not previously possible due to the embryonic lethality caused by the KO of this gene.

Service Details 

Service PackagePriceEst. Delivery Time
Full Build $4,075 and up6 Weeks and up
Candidate Lines $2,554 and up3 Weeks and up
Custom Injection Mix $995 and up1 - 2 Weeks

Publications

  • Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning & reduces neurodegeneration.

    Veronica H. Ryan, Theodora Myrto Perdikari, Mandar T. Naik, Camillo F. Saueressig, Jeremy Lins, Gregory L. Dignon, Jeetain Mittal, Anne C. Hart, Nicolas L. Fawzi. bioRxiv. March 18, 2020.

  • Tensin regulates pharyngeal pumping in Caenorhabditis elegans.

    Aaron N.Bruns, Su Hao Lo. Biochemical and Biophysical Research Communications
    Volume 522, Issue 3, 12 February 2020, Pages 599-603.

  • TRAIP drives replisome disassembly and mitotic DNA repair synthesis at sites of incomplete DNA replication.

    Remi Sonneville, Rahul Bhowmick, Saskia Hoffmann, Niels Mailand, Ian D Hickson, Karim Labib. eLife 2019;8:e48686.

  • DOT1L complex suppresses transcription from enhancer elements and ectopic RNAi in Caenorhabditis elegans.

    Esse R, Gushchanskaia ES, Lord A, Grishok, A. RNA. 2019.  

  • NEW FINDING A CRISPR/Cas9-generated cdc-7 loss of function mutation does not cause temperature-dependent fertility defects.

    Heather Currey and Nicole Liachko. microPublication Biology, 2019.

  • A Role in Apoptosis Regulation for the rad-51 Gene of Caenorhabditis elegans.

    Germoglio M, Adamo A. Genetics. 2018 Aug;209(4):1017-1028.

  • WAH-1/AIF regulates mitochondrial oxidative phosphorylation in the nematode Caenorhabditis elegans.

    Troulinaki K, Büttner S, Marsal Cots A, Maida S, Meyer K, Bertan F, Gioran A, Piazzesi A, Fornarelli A, Nicotera P, Bano D. Cell Death Discov. 2018 Jan 29;4:2.

  • Loss of function CHCHD10 mutations in cytoplasmic TDP-43 accumulation and synaptic integrity.

    Woo JA, Liu T, Trotter C, Fang CC, De Narvaez E, LePochat P, Maslar D, Bukhari A, Zhao X, Deonarine A, Westerheide SD, Kang DE. Nat Commun. 2017 Jun 6;8:15558.

  • Eyes shut homolog is required for maintaining the ciliary pocket and survival of photoreceptors in zebrafish.

    Yu, M; Liu, Y; Li, J; Natale, BN; Cao, S; Wang, D; Amack, JD; Hu, H. Biol Open. 2016 Nov 15;5(11):1662-1673.

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