Generation of Two Noradrenergic-Specific Dopamine-Beta-Hydroxylase-FLPo Knock-In Mice Using CRISPR/Cas9-Mediated Targeting in Embryonic Stem Cells
| Autor: | Jenny J. Sun, Russell S. Ray |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
lcsh:Medicine Dopamine beta-Hydroxylase Mice Catecholamines 0302 clinical medicine Antibiotics Medicine and Health Sciences Recombinase CRISPR Inbreeding Gene Knock-In Techniques lcsh:Science Genetics Multidisciplinary Antimicrobials Homozygote Brain Drugs Gene targeting Neomycins Cell biology Gene Targeting DNA Nucleotidyltransferases Anatomy Brainstem Sequence Analysis Research Article RNA Guide Kinetoplastida Genotyping Heterozygote Mice Transgenic Biology Research and Analysis Methods Microbiology 03 medical and health sciences Sequence Motif Analysis Microbial Control Gene knockin Animals Site-specific recombinase technology Molecular Biology Techniques Sequencing Techniques Molecular Biology Embryonic Stem Cells Pharmacology Cas9 lcsh:R Biology and Life Sciences Vector Cloning Embryonic stem cell 030104 developmental biology Genetic Loci Artificial Genetic Recombination lcsh:Q CRISPR-Cas Systems Homologous recombination 030217 neurology & neurosurgery Cloning Brain Stem |
| Zdroj: | PLoS ONE, Vol 11, Iss 7, p e0159474 (2016) PLoS ONE |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0159474 |
| Popis: | CRISPR/Cas9 mediated DNA double strand cutting is emerging as a powerful approach to increase rates of homologous recombination of large targeting vectors, but the optimization of parameters, equipment and expertise required remain barriers to successful mouse generation by single-step zygote injection. Here, we sought to apply CRISPR/Cas9 methods to traditional embryonic stem (ES) cell targeting followed by blastocyst injection to overcome the common issues of difficult vector construction and low targeting efficiency. To facilitate the study of noradrenergic function, which is implicated in myriad behavioral and physiological processes, we generated two different mouse lines that express FLPo recombinase under control of the noradrenergic-specific Dopamine-Beta-Hydroxylase (DBH) gene. We found that by co-electroporating a circular vector expressing Cas9 and a locus-specific sgRNA, we could target FLPo to the DBH locus in ES cells with shortened 1 kb homology arms. Two different sites in the DBH gene were targeted; the translational start codon with 6-8% targeting efficiency, and the translational stop codon with 75% targeting efficiency. Using this approach, we established two mouse lines with DBH-specific expression of FLPo in brainstem catecholaminergic populations that are publically available on MMRRC (MMRRC_041575-UCD and MMRRC_041577-UCD). Altogether, this study supports simplified, high-efficiency Cas9/CRISPR-mediated targeting in embryonic stem cells for production of knock-in mouse lines in a wider variety of contexts than zygote injection alone. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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