yruvate dehydrogenase kinase-4 (PDK-4) is a widely expressed isoform of PDK and is responsible for inhibiting pyruvate ehydrogenase (PDH) complex activity via phosphorylation. When PDK-4 is active, the PDH complex is in its phosphorylated ate and pyruvate cannot be converted into acetyl-CoA for entry into the citric acid cycle. Instead, in gluconeogenic tissues like ver and kidney, pyruvate is channeled to support gluconeogenesis. Conversely, when PDK-4 is inactive, the PDH complex eadily converts pyruvate into acetyl-CoA. researcher wants to determine the role of PDK-4 on blood glucose homeostasis. To accomplish this, she proposes to generate PDK-4 knockout mouse using CRISPR technology. To create the knockout mouse, she must first generate PDK-4 knockout mbryos to implant into pseudopregnant female mice. rrange the steps to outline the process by which she will generate the PDK-4 knockout embryos. First step Last step Answer Bank Generate Cas9 mRNA and sgRNA specific to the target sequence using in vitro transcription. Isolate single cell mouse embryos and inject them with Cas9 mRNA and sgRNA. Identify a protospacer-adjacent motif (PAM) adjacent to PDK-4 in the genome. Determine the DNA sequence of the region of the genome containing PDK-4.
yruvate dehydrogenase kinase-4 (PDK-4) is a widely expressed isoform of PDK and is responsible for inhibiting pyruvate ehydrogenase (PDH) complex activity via phosphorylation. When PDK-4 is active, the PDH complex is in its phosphorylated ate and pyruvate cannot be converted into acetyl-CoA for entry into the citric acid cycle. Instead, in gluconeogenic tissues like ver and kidney, pyruvate is channeled to support gluconeogenesis. Conversely, when PDK-4 is inactive, the PDH complex eadily converts pyruvate into acetyl-CoA. researcher wants to determine the role of PDK-4 on blood glucose homeostasis. To accomplish this, she proposes to generate PDK-4 knockout mouse using CRISPR technology. To create the knockout mouse, she must first generate PDK-4 knockout mbryos to implant into pseudopregnant female mice. rrange the steps to outline the process by which she will generate the PDK-4 knockout embryos. First step Last step Answer Bank Generate Cas9 mRNA and sgRNA specific to the target sequence using in vitro transcription. Isolate single cell mouse embryos and inject them with Cas9 mRNA and sgRNA. Identify a protospacer-adjacent motif (PAM) adjacent to PDK-4 in the genome. Determine the DNA sequence of the region of the genome containing PDK-4.
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
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Transcribed Image Text:### Pyruvate Dehydrogenase Kinase-4 (PDK-4) and Glucose Homeostasis
**Overview:**
Pyruvate dehydrogenase kinase-4 (PDK-4) is an isoform of PDK known for its role in inhibiting the pyruvate dehydrogenase (PDH) complex through phosphorylation. In active form, it prevents the conversion of pyruvate to acetyl-CoA, affecting the citric acid cycle. This regulation impacts gluconeogenic tissues like the liver and kidney. When inactive, it facilitates pyruvate conversion, enhancing acetyl-CoA production.
**Research Objective:**
A researcher aims to explore PDK-4's role in blood glucose homeostasis by creating a PDK-4 knockout mouse using CRISPR technology. This involves creating embryos lacking PDK-4 for implantation in pseudopregnant female mice.
**Experimental Steps:**
The researcher must follow these steps to generate PDK-4 knockout embryos using CRISPR technology:
1. **Determine the DNA sequence** of the region containing PDK-4.
2. **Identify a protospacer-adjacent motif (PAM)** adjacent to PDK-4 in the genome.
3. **Generate Cas9 mRNA and sgRNA** specific to the target sequence using *in vitro* transcription.
4. **Isolate single cell mouse embryos** and inject them with Cas9 mRNA and sgRNA.
These steps outline the process of creating the PDK-4 knockout embryos, starting with DNA sequencing and ending with embryo injection.
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