VPS29 결손의 기능적 분석을 통한 배아 발달 및 Retromer 안정성에서의 역할 규명 = Functional analysis of VPS29 deficiency reveals its role in embryonic development and retromer stability

다국어 초록 (Multilingual Abstract)

Intracellular protein trafficking is essential for normal development and tissue homeostasis, and the retromer complex plays a central role in regulating retrograde transport from endosomes to the trans-Golgi network as well as membrane protein recycling. VPS29 is a core component of the retromer complex and has been implicated in maintaining its structural stability; however, its in vivo function has not been fully elucidated. In this study, we generated a Vps29-deficient mouse model using CRISPR/Cas9-mediated genome editing and investigated the role of VPS29 in embryonic development and retromer complex stability. Genotypic analysis revealed that homozygous Vps29 knockout (Vps29−/−) mice were not observed at birth, whereas Vps29−/− embryos were detected at early developmental stages but were progressively lost during embryogenesis. Morphological analysis demonstrated that Vps29−/− embryos exhibited reduced somite formation and delayed overall development. Molecular analyses further showed that VPS29 deficiency was accompanied by decreased protein expression of other retromer core components, VPS26 and VPS35. In addition, heterozygous Vps29+/− mice also displayed developmental delay and gross anatomical abnormalities. Taken together, these findings suggest that VPS29 plays an important role in maintaining retromer complex stability and supporting normal embryonic development and survival in vivo. This study provides fundamental insights into the role of VPS29 and retromer-mediated trafficking in developmental processes and tissue homeostasis.

목차 (Table of Contents)

• (Abstract)
• Ⅰ.INTRODUCTION 1
• Ⅱ. MATERIALS AND METHODS 6
• 1. Animals and ethics approval 6
• 2. CRISPR/Cas9-mediated generation of Vps29 knockout mouse 6
• 3. Genotyping of Vps29 knockout mouse 7
• 4. Embryo collection and genotype distribution analysis 8
• 5. RNA extraction and RT-PCR analysis 9
• 6. Protein extraction and western blot analysis 10
• 7. Statistical analysis 10
• Ⅲ. RESULTS 11
• 1. Generation of Vps29–deficient mouse 11
• 2. Genotyping PCR analysis of Vps29 knockout mouse 13
• 3. Genotypic distribution of newborn mouse from heterozygous intercross 13
• 4. Survival and fertilized embryo formation analysis 14
• 5. Genotypic validation and VPS29 protein expression analysis in Vps29-deficient mouse 15
• 6. VPS29 deletion reduces retromer complex expression analysis 16
• 7. Genotypic distribution of early embryos 17
• 8. VPS29 deletion impairs embryonic development 18
• 9. VPS29 deletion causes gross morphological abnormalities in mouse 20
• Ⅳ. DISCUSSION 21
• 1. VPS29 deletion causes embryonic lethality 21
• 2. VPS29 deletion imparis embryonic development 22
• 3. VPS29 deficiency destabilizes the retromer complex 22
• 4. Phenotypic abnormalities in Vps29 heterozygous mouse 23
• 5. Significance and limitations of the study 23
• Ⅴ.CONCLUSION 25
• Ⅵ.REFERENCE 26