Principle of PTSelect™ technology in establishment of stable cell
population
The expression of PTSelect™-siRNA serves as a proxy for GOI expression,
eliminating the need for drug resistance gene(s). To select stable
cells, CD4/siRNA mRNA, which contains RNA sequences complementary to the
PTSelect™-siRNA is electroporated into the cells three days after the
transfection of GOI. mRNA electroporation is highly effective with
~99% transfection efficiency (Supplementary Figure 2a).
In cells that express the GOI and therefore produce the PTSelect™-siRNA,
CD4 expression is downregulated, preventing CD4 expression on the cell
surface. Conversely, cells that do not contain the GOI, do not produce
PTSelect™-siRNA and consequently express CD4 on the cell surface (Figure
1c). Maximum CD4 expression from the electroporated mRNA occurs at
~10 hours after electroporation (Supplementary Figure
2b). To separate CD4-positive (no GOI) from CD4-negative (expressing
GOI) cells, 6 hours after electroporation, the cells are incubated with
anti-CD4-conjugated magnetic beads. Exposing the cells to a magnetic
field, the GOI-negative cells attached to the beads adhere to the inner
walls of the tube, while the CD4-negative (GOI positive) cells remain
suspended and are removed (Figure 1d). From electroporation of CD4/siRNA
mRNA to separation constitutes one round of depletion and is repeated on
days 3, 7 and 10 after initial transfection of GOI to obtain an enriched
pool of stable cells.