Anti-rdl and anti-mglutr1 receptors antibody testing in honeybee brain sections using crispr-cas9

Irina Sinakevitch, Zev Kurtzman, Hyun G. Choi, David Arturo Ruiz Pardo, Romain A. Dahan, Nathaniel Klein, Branimir Bugarija, Erik Wendlandt, Brian H. Smith

Research output: Contribution to journalArticlepeer-review

Abstract

Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing technique widely used in studies of gene function. We use this method in this study to check for the specificity of antibodies developed against the insect GABAA receptor subunit Resistance to Dieldrin (RDL) and a metabotropic glutamate receptor mGlutR1 (mGluRA). The antibodies were generated in rabbits against the conjugated peptides specific to fruit flies (Drosophila melanogaster) as well to honeybees (Apis mellifera). We used these antibodies in honeybee brain sections to study the distribution of the receptors in honeybee brains. The antibodies were affinity purified against the peptide and tested with immunoblotting and the classical method of preadsorption with peptide conjugates to show that the antibodies are specific to the corresponding peptide conjugates against which they were raised. Here we developed the CRISPR-Cas9 technique to test for the reduction of protein targets in the brain 48 h after CRISPR-Cas9 injection with guide RNAs designed for the corresponding receptor. The CRISPR-Cas9 method can also be used in behavioral analyses in the adult bees when one or multiple genes need to be modified.

Original languageEnglish (US)
Article numbere59993
JournalJournal of Visualized Experiments
Volume2020
Issue number155
DOIs
StatePublished - Jan 2019

Keywords

  • CRISPR-Cas9
  • Conjugated antibodies controls
  • GABA receptor
  • Issue 155
  • MGlutR1
  • Metabotropic glutamate receptors
  • Neuroscience
  • RDL

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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