Note taking worksheet (7)

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Journal Article Note Taking Worksheet Mostly comments should be in your own words. Copy and paste is discouraged. If quotes are used be sure to add quotation marks. This template is designed to help you comprehend and evaluate the article. Add sufficient detail to allow you to participate fully in the discussion during class. (Bold categories require your judgment and are not necessarily free of opinion [i.e. they require your assessment). Citation: First Author, Date of publication, Title, Journal R.Hu, 2017, Generation of FGF5 knockout sheep via the CRISPER/Cas9 system, American Society of Animal Science Executive summary (in 6 sentences or fewer, summarize this article as if you were explaining it to your parents): This article explores the application of the CRISPER/Cas9 gene editing technology to produce sheep with a targeted knockout of the fibroblast growth factor 5 (FGF5) gene. FGF5 is known to play a crucial role in regulating hair length in mammals, making it a potential target for genetic modification to influence wool characteristics in sheep. In the study they introduced targeted mutations in the FGF5 gene of sheep embryos. Following the gene editing process, the researchers successfully generated FGF5 knock out sheep. The study contributes to the growing body of research on CRISPER/Cas 9 mediated genetic modifications in livestock and highlights the possibilities of using this technology to enhance desirable traits in animals for agricultural purposes. Objective/question (what is/are the author’s objective[s], why was the experiment conducted): To employ the CRISPER/Cas9 gene editing system to generate FGF5 knockout sheep, aiming to investigate the impact of fibroblast growth factor 5 (FGF5) gene distribution on wool characteristics Hypothesis (what is the author’s hypothesis, if not explicitly stated by the authors infer one from the context and design of the study): That the targeted knockout of the FGF5 gene in sheep using the CRISPER/Cas9 system will result in significant alterations in wool characteristics Methodology (bullet list of what was done, include treatments, # of animals, etc):  Identify target sites within the FGF5 gene for knockout  Design guide RNA sequences for CRISPER targeting  Construct CRISPER/Cas9 plasmids containing the guide RNA sequences and Cas9 nuclease  Validate the efficiency and specificity of the CRISPER/Cas 9 constructs using in vitro assays  Transfect cultured sheep cells with crisper/cas9 plasmids  Analyze genomic DNA from transfected cells to confirm the introduction of targeted mutations  Obtain healthy sheep embryos for the gene editing procedure  Culture the embryos in vitro to the blastocyst stage for optimal CRISPER/Cas9 constructs into the cytoplasm of cultured sheep embryos at the blastocyst stage  Optimize injection conditions for precision and minimal damage to embryos

 Select viable embryos with successful CRISPER/Cas9 integration  Transfer edited embryos into recipient ewes for gestation  Collect tissue samples from newborn lambs  Extract genomic DNA from tissue samples for genotyping analysis  Employ PCR and sequencing techniques to confirm the presence of FGF5 gene mutations in the offspring  Monitor and document the growth and development of FGF5 knockout sheep  Assess wool characteristics such as length and texture in comparison to wild-type sheep Results (bullet list of findings, main outcomes):  155 1 cell embryos were collected  Cas9 mRNA and sgRNA were pooled and microinjected into the cytoplasm of embryos  100 injected 1cell embryos were transferred into 53 surrogate females  1 recipients were pregnant on day 30 to 35 after the embryo transfer  18 lambs were born including 5 twins  The genomic modification occurred in 3 founder animals including 2 females and a male  The FGF5 mutated individuals presented in their genomes various amounts of integration, the integration efficiency was 15.8% Discussion (were the objectives met, is there speculation, are results statistically and biologically significant, was discussion clear and relevant, ) Yes their objective was met, even though they had 3 out of the 18 carry the gene they still had a successful efficiency. Conclusion (summarize key points, do results support conclusions): In conclusion this study was successful it demonstrated the application of the CRISPER/Cas9 gene editing system for generating FGF5 knockout sheep, shedding light on the intricate relationship between the FGF5 gene and wool characteristics in sheep. Important Figures and/or Tables (brief description of why this table/figure is important to the paper): In figure 2 it is showing the changes in the biological function of FGF5 in 1 month old founders, this helps us understand what is going on with that gene inside the animal while it is growing In figure 1 it is showing the generations of FGF5 knockout sheep by Cas9 mediated modifications, this helps us understand what they did from previous studies to what they are doing now in the study Critical analysis and reflection (what do these data mean in the real world, what implications can you think of, are further experiments necessary): This means we can successfully generate FGF5 knockout sheep using the CRISPER/Cas9 modifications some other things I thought of when reading this is can we use this in other animals that are used for the production of their hair like llamas and some other mammals and see if they get the same results or get different results

Questions (list your questions here, include concepts, ideas, details, etc you didn’t understand that could be discussed in class): What would happen if they tried this on different animals would they get the same results or get different ones?

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