LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

normal female, pups were screened for the presence of EGFP-f by PCR amplification
from genomic DNA. 14-3-3ε levels were determined in the EGFP-f positive mice by
reverse transcriptase coupled PCR (RT-PCR). The levels of knockdown were established
by comparing the levels of 14-3-3ε mRNA to that of the housekeeping gene GAPDH.
Three animals that showed a decreased level of 14-3-3ε died within 160 days of birth, as
compared to control mice. A decrease in body weight upon downregulation of 14-3-3ε
was also observed in the knockdown mice.
It was reported earlier that a mice that are heterozygous or homozygous null for 14-3-3ε
have defects in brain development and neuronal migration (363). In the homozygous null,
most mice die just after birth. These results suggest that loss of 14-3-3ε leads to defects in
brain development. To determine if this phenotype was observed in our 14-3-3ε
knockdown mice, brain sections from wild type or knockdown mice were stained with
antibodies to 14-3-3ε and examined under the microscope. It was observed that there is a
decrease in staining for 14-3-3ε in brain sections derived from the knockdown mice as
opposed to sections derived from wild type mice. The defect in the cortical layers
observed was not significant.
The 14-3-3ε knockdown mice died about 160 days post birth. To determine the cause of
death in the knockdown mice, the mice were dissected and the internal organs examined.
All the mice had significantly enlarged spleens (splenomegaly).To determine the cause of
the splenomegaly and the alterations in the other internal organs, an immunohistochemical
analysis performed. It was observed that the lungs, liver and kidneys of the 14-3-3ε
knockdown mice showed the presence of infiltrating cells of lymphoid origin as compared
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