Event Title

Analysis Of Danio Rerio (Zebrafish) Lamtor3 Crispr Knockouts And The Effects On Embryotic Yolk Digestion And Pigmentation.

Location

Lobby in front of Auditorium

Start Date

19-4-2019 11:00 AM

Department

Biology

Description

Lamtor 3 is part of pentameric complex, known as the Ragulator complex, that is located on the lysosomal membrane and plays a role in the amino acid activation of the mTORC1 pathway. The mTORC1 pathway is a growth regulator that uses the availability of nutrients to activate a variety of metabolic processes that are responsible for cell growth and proliferation. The yolk serves as the main reservoir of nutrients for metabolic processes in early embryotic development. Using CRISPR Cas9 technique, a targeted double strand break was induced in the lamtor3 gene. Yolk digestion was observed and photographed using a Zeiss Axioplan microscope and yolk measurements were obtained by using Image J software. A paired-two tail T-test was conducted using excel. Yolk sizes were measured at 3 days post fertilization (dpf) and at 6 days post fertilization (dpf). At 3 dpf, there was not a significant difference in yolk sizes, but at 6 dpf there was a statistical difference in yolk measurements. The yolk sizes of the wild type controls were larger at 6 dpf. The working hypothesis of this experiment was that if lamtor 3 was inactivated and the Ragulator complex was inhibited, it would not be able to localize and activate the mTORC1 pathway. With mTOR inhibited, vital metabolic processes would not take place and utilization of yolk nutrients would be less, resulting in a larger yolk. The basis of this hypothesis was formed by rapamycin experiments that were conducted that inhibited the mTORC1 pathway. Yolk sizes were observed to be larger in developing embryos when mTORC1 was inhibited along with other developmentally abnormalities. Because lamtor3 is part of a pentameric structure, it appears that there might have been some compensation from the other lamtor subunits. This might have caused the need for additional nutrients or have caused the Ragulater to function less efficiently, ultimately depleting the yolk nutrients more quickly, resulting in a smaller yolk. Interestingly, one of the lamtor3 knockouts expressed a drastic phenotype mutation. Currently, using a mutagenesis detection kit, we are trying to assess the efficacy of the CRISPR Cas9 knockouts, and if the phenotype mutation occurred in the lamtor 3 gene region. The mutant has survived to adulthood and weekly crosses are being performed to see if the offspring also carry the mutant phenotype. Further research is needed to determine the cause of the differences in yolk size and to determine the cause of the mutant phenotype.

Comments

Jorge Cantu is the faculty sponsor of this poster.

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Apr 19th, 11:00 AM

Analysis Of Danio Rerio (Zebrafish) Lamtor3 Crispr Knockouts And The Effects On Embryotic Yolk Digestion And Pigmentation.

Lobby in front of Auditorium

Lamtor 3 is part of pentameric complex, known as the Ragulator complex, that is located on the lysosomal membrane and plays a role in the amino acid activation of the mTORC1 pathway. The mTORC1 pathway is a growth regulator that uses the availability of nutrients to activate a variety of metabolic processes that are responsible for cell growth and proliferation. The yolk serves as the main reservoir of nutrients for metabolic processes in early embryotic development. Using CRISPR Cas9 technique, a targeted double strand break was induced in the lamtor3 gene. Yolk digestion was observed and photographed using a Zeiss Axioplan microscope and yolk measurements were obtained by using Image J software. A paired-two tail T-test was conducted using excel. Yolk sizes were measured at 3 days post fertilization (dpf) and at 6 days post fertilization (dpf). At 3 dpf, there was not a significant difference in yolk sizes, but at 6 dpf there was a statistical difference in yolk measurements. The yolk sizes of the wild type controls were larger at 6 dpf. The working hypothesis of this experiment was that if lamtor 3 was inactivated and the Ragulator complex was inhibited, it would not be able to localize and activate the mTORC1 pathway. With mTOR inhibited, vital metabolic processes would not take place and utilization of yolk nutrients would be less, resulting in a larger yolk. The basis of this hypothesis was formed by rapamycin experiments that were conducted that inhibited the mTORC1 pathway. Yolk sizes were observed to be larger in developing embryos when mTORC1 was inhibited along with other developmentally abnormalities. Because lamtor3 is part of a pentameric structure, it appears that there might have been some compensation from the other lamtor subunits. This might have caused the need for additional nutrients or have caused the Ragulater to function less efficiently, ultimately depleting the yolk nutrients more quickly, resulting in a smaller yolk. Interestingly, one of the lamtor3 knockouts expressed a drastic phenotype mutation. Currently, using a mutagenesis detection kit, we are trying to assess the efficacy of the CRISPR Cas9 knockouts, and if the phenotype mutation occurred in the lamtor 3 gene region. The mutant has survived to adulthood and weekly crosses are being performed to see if the offspring also carry the mutant phenotype. Further research is needed to determine the cause of the differences in yolk size and to determine the cause of the mutant phenotype.