Digital Commons @ Shawnee State University - Celebration of Scholarship: Using CRISPR-Cas9 Techniques to Investigate Chick Limb Development
 

Thursday, 4/7/2022

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University

Shawnee State University

Major

Biology

Student Type

Undergraduate Student

Presentation Types

Oral Group Presentation (Live)

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 and Animal Subjects

no

IRB or IACUC Approval

no

Faculty Mentor Name

Dr. Kimberly Inman

Faculty Mentor Title

Chairperson and Associate Professor of Biology

Faculty Mentor Department

Natural Sciences

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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.