Thursday, 4/7/2022
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University
Shawnee State University
Major
Biology
Presentation Types
Oral Group Presentation
Keywords:
limbless, chick, CASPR-Cas9, chick
Abstract
CRISPR-Cas9 is used to investigate gene(s) underlying the limbless (ll) chick mutant phenotype. Guide RNA (gRNA) is used to bind to a specific DNA target sequence and the Cas9 enzyme facilitating specific cutting of DNA leading to decreased gene expression. The ll mutation has been mapped to an ~2.6 Mb region of chromosome 2. Genes in the targeted region suspected to have a role in limb development include: SP8 (expressed in limb ectoderm), Twist1 (promotes bone health and proliferation/lifespan of mesenchymal cells), and NUPL2 (regulates blood vessel development, smooth muscle contraction and hypoxia-related signaling pathways.) Target sequences for gRNAs will be identified using an online tool and synthesized commercially. Double stranded oligos will be ligated into the U6.3>gRNA.f+e plasmid and then co-electroporated with the pCAGG>nls-hCas9-nls-GFP plasmid into HH14 chick forelimb fields. Embryos will be incubated for 24-48 hours to examine limb development compared to the controls.
Human Subjects
no
IRB Approval
no
Faculty Mentor Name
Dr. Kimberly Inman
Faculty Mentor Title
Chairperson and Associate Professor of Biology
Faculty Mentor Academic Department
Natural Sciences
Recommended Citation
Jenkins, Sydnie; Coriell, Collin; and Heimbach, Abraham, "Using CRISPR-Cas9 Techniques to Investigate Chick Limb Development" (2022). Celebration of Scholarship. 5.
https://digitalcommons.shawnee.edu/cos/2022/day4/5
Using CRISPR-Cas9 Techniques to Investigate Chick Limb Development
CRISPR-Cas9 is used to investigate gene(s) underlying the limbless (ll) chick mutant phenotype. Guide RNA (gRNA) is used to bind to a specific DNA target sequence and the Cas9 enzyme facilitating specific cutting of DNA leading to decreased gene expression. The ll mutation has been mapped to an ~2.6 Mb region of chromosome 2. Genes in the targeted region suspected to have a role in limb development include: SP8 (expressed in limb ectoderm), Twist1 (promotes bone health and proliferation/lifespan of mesenchymal cells), and NUPL2 (regulates blood vessel development, smooth muscle contraction and hypoxia-related signaling pathways.) Target sequences for gRNAs will be identified using an online tool and synthesized commercially. Double stranded oligos will be ligated into the U6.3>gRNA.f+e plasmid and then co-electroporated with the pCAGG>nls-hCas9-nls-GFP plasmid into HH14 chick forelimb fields. Embryos will be incubated for 24-48 hours to examine limb development compared to the controls.