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University
Shawnee State University
Major
Biology - Biomedical Sciences
Presentation Types
Oral Group Presentation
Keywords:
chick limb, CRISPR, transformation, embryos
Abstract
CRISPR-Cas9 is used to investigate gene(s) underlying the limbless (ll) chick
mutant phenotype. 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, Twist1, and NUPL2. Target sequences for guide RNAs (gRNAs) were identified using an online tool and synthesized commercially. Double stranded oligos were ligated into the U6.3>gRNA.f+e plasmid. The newly ligated plasmid was transformed into competent cells, plated onto LB-ampicillin plates, and expanded in liquid culture. A mini prep was performed to isolate plasmid DNA to allow the gRNA production in cells of the chick embryo. Future plans include co-electroporating the oligo containing U6.3>gRNA.f+e plasmid with 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
Kimberly Inman
Faculty Mentor Title
Dean of Arts and Sciences
Faculty Mentor Academic Department
Natural Sciences
Recommended Citation
Jenkins, Sydnie and Coriell, Collin, "Using CRISPR-Cas9 Techniques to Investigate Chick Limb Development" (2023). Celebration of Scholarship. 13.
https://digitalcommons.shawnee.edu/cos/2023/Day5/13
Using CRISPR-Cas9 Techniques to Investigate Chick Limb Development
CRISPR-Cas9 is used to investigate gene(s) underlying the limbless (ll) chick
mutant phenotype. 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, Twist1, and NUPL2. Target sequences for guide RNAs (gRNAs) were identified using an online tool and synthesized commercially. Double stranded oligos were ligated into the U6.3>gRNA.f+e plasmid. The newly ligated plasmid was transformed into competent cells, plated onto LB-ampicillin plates, and expanded in liquid culture. A mini prep was performed to isolate plasmid DNA to allow the gRNA production in cells of the chick embryo. Future plans include co-electroporating the oligo containing U6.3>gRNA.f+e plasmid with 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.