Oral dual-therapy for prevention of obesity

Probiotics Team

OUTLINE:
we express our target genes (8 in total, 4 controls and 4 modified) in
1. E.coli expression vector pUC57 → commonly used vector, for stock storage & quantification
2. customised vector pTRKH3 in E.coli → for quantification
3. customised vector pTRKH3 in Lactobacillus caesi → for expression

VERIFICATION:
western blot → protein products
culture hepatocytes and adipocytes to perform cell assay → test if the product really help prevent obesity or lower fat content

miRNA Team

1. Generation of stable cell line producing adipose-targeted exosome (with 3 mi-RNA inside)
- Cell: HEK293
- Plasmid for transfection:


- Negative control:

- After transfection, screen for stable transfectants using puromycin
- Freeze some stocks in liquid nitrogen just in case


2. Characterization of the exosome
- western blot of FLAG tag in cell lysates and in supernatant
- If FLAG can be detected, you can purify exosome from cell supernatant and the exosome can be visualized under electron microscopy


3. Functional analysis on adipocytes
- Differentiate mouse stromal vascular cell line to mature adipocytes. Consider to differentiate them to beige adipocytes
- After differentiation, treat the adipocytes with the exosome. You can either use cell supernatant or purified exosome (maybe supernatant at this stage is good enough)
- To test the effect of the exosome, harvest the adipocytes and measure the mRNA expressions of your target genes first. Additionally, you can check the anti-inflammatory, pro-beiging, and anti-senescence effects, by measuring the markers genes for these three pathways. Please ask Xuemei and Dr. Zhao for those marker genes and primers for inflammation and beiging
- If all went smoothly, additional functional assays can be considered, such as seahorse bioanalysis (to measure the energy expenditure rate of the adipocytes), and beta-galactosidase assay (senescence marker)