In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.

2. IN VIVO ANALYSIS OF AUTOPHAGY IN RESPONSE
TO NUTRIENT STARVATION USING TRANSGENIC
MICE EXPRESSING A FLUORESCENT
AUTOPHAGOSOME MARKER
Research by:Yoshinori ohsumi and his team.
Presentaton by khalid zaffar

3. ABSTRACT
Macro autophagy mediate the bulk degradation of cytoplasmic component.
It account for the degradation of of most long lived cytoplasmic component.
To understand where and when the autophagy occur in vivo we generate transgenic mice by expressing GFP fused with
LC3.
Fluorescence microscopy analysis the function of autophagy during starvation period different from one tissue to other
and as well as one organ to the other.
The study revealed that autophagy is organ dependent and is not restricted to starvation response.
The transgenic mouse model is useful tool to study mammalian autophagy.

4. INTRODUCTION
Eukaryoute have to major protein degradation system with in the cell. One is ubiquitin proteasome system and the other is
lysosomal system
In lysosomal the degradation of exogenous material and plasma membrane protein is mediated by endocytosis and
phagocytosis.
Where is cytoplasmic component is achieved by autophagy or autophagocytosis. There is three type of autophagy from
those will study only about macroautophgy.

5. Formation of autophagolysosome

6. MATERIAL AND METHOD
Antibodies.
Polyclonal anti –GFP Antibody for immunoblotting and alexa fluro 568 goat
anti rabbit lgG antibody
Rabbit anti mouse keratin antibody

7. CONSTRUCTION OF PLASMID
• 1.8 kbp dna fragment
• Rat lcd Cdna fuced with EGFP
• Insertion in cytomegalovirus in downstream
• Generate PCAG-GFP-LC3

8. GENENRATION OF F9 STABLE
TRANSFORMANT
• F9 tetracarcinoma cell
• DMEM containing fetal serum

9. GENERATION OF TRANSGENIC MICE
• 3.4 kbp xhol and spel fragment
• Injected in fertilized egg
• Transfer in pseudo pregnant foster femal.
• Through PCR Out of 60 eight were positive for transgene
• One of transgenic line use here

10. SOME MATERIAL ALSO USED HERE
IMMUNO BLOTTING.
FLUORESCENCE MICROSCOPY

11. Gfp-lc3 as marker for autophagic membranes.

12. EXPRESSION OF GFP-LC3 TRANSGENIC MICE THROUGH SDS PAGE

13. liver

14. muscles

15. PANCREASE

16. KIDNEY

17. OVERVEIW

18. DISCUSSION
We have monitor autophagy in vivo
Using gfp-lc3 has several advantage
Its simple and quick procedure it enable us to analyse large number
Sample.
2nd labelling gfp-lc3 is specific for identification of autophagy
This accurate for monitoring autophagy
Over expression of marker gene did not affect endogenious autophagic procces.
We can observe it not only the cell but also in tissue

19. conclusion
We have develop a novel method to monitor autophagy in the mouse.
its simple quick and accurate. this gives us analysis of autophagy in response to
starvation in accurate manner.
This system can be utilized to study a number of yet unsolved problem regarding
autophagy like significance of autophagy in disease cell death cell degeneration and
development etc.

21. ‫شکریھ‬