Methods used in the Kanarek Lab
Metabolite profiling by mass spectrometry
We have two in-house mass spectrometers (Thermo Q-Exactive) that allow us to detect folate, nucleotides, amino acids, sugars and other relevant metabolites in cells, tissues, tumors and whatnot.
CRISPR/Cas9 functional genomic screens
We currently use functional genomics to reveal new components of the cellular response to folate deprivation.
In the future we will apply this powerful tool for the discovery of new biology in other biological questions in folate metabolism in cancer, autoimmune diseases and development.
CRISPR-based individual gene knockouts
We use the most up-to-date genetic tools available in order to study folate in a rigorous way. We really like to perturb genes when we study them so we can have a powerful handle to assess the function of the studied genes.
In vivo tumor models
In order to identify new folate-related vulnerabilities of tumors, and to study whole-body homeostasis of folate in tumor-bearing organisms, we use models that are as close as possible to the human disease, such as orthotopic xenografts and patient-derived xenografts.
We already found through our CRISPR/Cas9-based screens some interesting genes that are likely to play key roles in the cellular response to folate deprivation. Now we are studying these genes in vivo (and others will follow in the future) by genetic perturbations in specific tissues that are most relevant for our research.
See figure below.
As part of our research we are required to maintain expertise in cutting-edge molecular biology tools: cloning, gene targeting, mutagenesis, and synthetic genes are just some of the tools we need to apply in order to robustly study cellular and organismal regulation and sensing of folate.
We study signal transduction, transcription, apoptosis, DNA damage, and other cellular processes that require comprehensive biochemical work.
Genes that were found to have a role in the survival of cells in conditions of low folate by our functional genomic screens will be targeted in vivo.
We will implement CRISPR/Cas9 technology to generate transgenic models and to study the physiological function of the candidate genes in folate homeostasis in the whole animal in health and in several pathological conditions.