BC Files Genetic

Re: your memo/latest results

Sir:

We take your criticisms very seriously but please recognize the enormity of our task here. We have already silenced 43 genes using the most advanced siRNA and shRNA technology; we work tirelessly throughout the night and sometimes into the morning, depending on class schedules; and we continue to re-use the same student samples without complaint. All we ask is that you take other matters into consideration beyond our immediate cost-effectiveness.

We think you will be pleased to see our latest gene knockouts. Together, these genes are essential to:

• maintaining stability in undifferentiated cells
• suppressing the development of cancer cells
• promoting cell proliferation in contact with undifferentiated cells

I will let the results speak for themselves. I believe it is only a matter of time before we reach our goal.

Gene Silencing Study 104-A

Introduction

We have successfully knocked out the following 4 genes in blood samples by using short hairpin RNAs (shRNAs) for RNA interference (RNAi):

• TP53 tumor protein p53 (Li-Fraumeni syndrome)
• CXCL12 chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)
• TGFB1 transforming growth factor, beta 1 (Camurati-Engelmann disease)
• TNF tumor necrosis factor (TNF superfamily, member 2)

Each of these genes has an important halohelix function:

• TP53 could play a significant part in suppressing cancer cell development.
• CXCL12 promotes the rapid reproduction of hematopoietic progenitor cells in

connection with mesenchymal cells.

• TGFB1, a multifunctional peptide, manages proliferation, differentiation, and

other activities in several cell types.

• TNF encodes a cytokine that partly controls a broad range of biological processes

including cell proliferation, lipid metabolism, apoptosis and coagulation.

The silencing of three of the four genes had significant negative consequences, but did not immediately reveal any unexpected genetic activity.

Gene Gene Suppression Chart

All four genes displayed a significant reduction due to RNAi, particularly during the initial 24-hour phase.

Gene TP53

Gene CXCL12

Gene TGFB1

Gene TNF

Results

The silencing of genes in Study 104-A had serious effects in three out of four cases:

• TP53 alleles were disrupted by homologous recombination; we were also able to

identify 35 genes whose expression is correlated to TP53.

• Silencing of CXCL12 disrupted the directed migration of leukocytes, lymphocytes

and hematopoietic cells through CXCR4-expressing cells.

• Regarding TGFB1, basal secretion of IL-8 and MCP-1 both diminished (p<0.05)

but surprisingly, fibronectin grew (p<0.05) in TGF-1 silenced cells in comparison to cells that were transfected with non-specific siRNA.

• In TNF, changes in tissue levels were barely detectable.

In conclusion, all four genes can be successfully knocked out and will continue to be silenced in future studies.