Nature publication

1. Presentation by: S. Jaishree Reverian Habimana
(Oct. 12/2022) PhD Student/BMS Masters Student/BMS
Advisor: Prof. Jongkeun Seon Advisor: Prof. In Seok Jeong

2. • Tumour suppressor gene(1979)
• Unstable & degrades very quickly
• Mutated commonly during cancer
Function
• Regulation of cell cycle
• DNA Repair
• Apoptosis
p53 gene

3. Cell cycle Checkpoints

4. DNA damage
Triggers p53
gene expression
Increased p53
levels
Prevent cell
from entering S
phase of cell
cycle
Arrest of cell
cycle at G1
phase
Allows time for
the DNA repair
to take place
DNA repaired
P53 degrades
and cell cycle
continues
DNA not
repaired
Permanent
arrest /
Senescence
Process of p53 in DNA damage

5. p53 gene targeting
• p53 targeting vector constructed.
• Electroporated with ES cells. Selection in
G418 and FIAU (100 resistant colonies)
• Screened by PCR (Gene targeting specific
junction fragment)
• 2 colonies were positive

6. • Southern blot hybridisation – 3probes
• This concluded that homologous
recombination was achieved in the 2 ES cell
colonies.
Wt- Control DNA
1,2 – Cell clones confirmed by PCR
Confirmation of gene replacement

7. • ES cell clones microinjected into 3.5day old C57BL/6 blastocysts
• Implanted to pseudo pregnant F1 females (Offspring – Many Chimeric Mice)
p53 deficient mice

8. p53 deficient mice
• To assess the role of p53 in development.
• The homozygotes appeared to be normal both
morphologically and histologically.

9. 1-5 Lanes (Mice heterozygous for disrupted p53 allele)
6-7 Lanes (Mice homozygous for disrupted p53 allele)
8 Lane (Wild type Genotype)
Confirmation of p53 null allele
• Targeted the p53 was a null allele and normal p53 was not synthesised in tissues of
homozygous mice.

10. • Analysed total RNA from mouse fibroblasts
by northern blotting.
• RNA-PCR strategy.
Confirmation of p53 null allele

11. a- Protein Immunoblotting
2,5,8 lanes (specific amino terminus of p53 protein)
3,6,9 lanes (entire p53)
• Neither a truncated nor full length p53 is produced in homozygous mice.
b- Metabolic labelling
5,6,8,9 lanes (amino specific p53 protein)
Confirmation of p53 null allele

12. Tumour susceptibility
• To test the effects of the loss of p53 on tumour development.
• The original chimeras, heterozygote, homozygotes, and wild type (normal littermates)
were monitored by spontaneous neoplasms.
Mice No. of Mice Period Tumour
Wild type 95 Till the age of 9months No tumour
Chimeric mice 1 14months Osteogenic sarcoma,
ovarian choriocarcinoma
Chimeric mice 1 14months Leydig cell tumour- testis
Heterozygous Mice 2 of 96 5months
9months
Embryonal carcinoma
malignant lymphoma

13. Tumour susceptibility
• Total of 35 homozygote mice
were observed.
• 6 died by unknown causes
(4 – Non tumour deaths)
(2 – Obvious tumours)

14. Tumour susceptibility
a) Subcutaneous haemangiosarcoma
b) Sacral osteosarcoma
c) Subcutaneous undifferentiated sarcoma
d) Testicular gonodoblastoma
e) Leydig cell tumour- Testis
f) Embryonal carcinoma- Testis
g) Choriocarcinoma
h) Lymphoblastic malignant lymphoma
• Histopathological analysis of different tumours from p53 deficient mice.

15. Conclusion

16. Thank you