Gene Functionalization

Defined cDNAs are arrayed onto white or black-clear bottom 384 well TC treated [Greiner] plates. Using the CCS Packard MiniTrak, 20-62.5ng of cDNA are “spotted out” per well. The MiniTrak's unique MultiPosition Dispense Module (MPD) integrates a multi-position labware deck with highly precise dispense head positioning to facilitate reliable and accurate pipetting into 96- and 384- well microplates. Using p20 tips, volumes can range from 0.5-20 µL and can achieve accuracy of =1% CV across 96 wells and =1% plate to plate when dispensing 5µL of water or DMSO into liquid.

MiniTrak Systems incorporate high capacity Stackers, with each Stacker holding up to 50 SBS standard microplates. Before these cDNA libraries can be spotted out, cDNAs are first expanded in a high throughput fashion and miniprepped. Once prepped, each cDNA is normalized onto 96 well plates. At the same time, hundreds of plates are bar coded and labeled in order to facilitate separation after spotting and so that plates can be immediately sealed and stored at -80 c until needed for separation into sets. The process of spotting involves collapsing 4-96 well source plates into one 384 destination plate per round of the Minitrak run leaving the bottom 4x4 wells empty for controls. Normally 20-40 rounds are performed yielding as many identical copies. Our libraries have been completed with as many as 50+ runs to as few as 1 run, each run taking approximately 45 minutes. Once all the runs have been completed, the copies are separated into complete sets and stored at -80 C until needed for screening. On the day of screening, a set is pulled out of the freezer and allowed to thaw. Once thawed, controls are spotted out onto each plate and lipid-based transfection is then performed. Currently, we have an unprecedented collection of cDNA libraries consisting of: 1) 20K FGA set, 2) 11K set [human and mouse], 3) 16K Origene set [human] and 4) 10K siRNA set [human]. The bottom 4x4 of each 384-well plate is left empty for controls. Once the controls have been spotted out onto each plate, lipid-based transfection can be performed [retro-transfection].

The Titertek 384 MultiDrop, an instrument that dispenses to both 96 well and 384 well plates with volume ranges of 5 µL to 395 µL, and 5 µL to 100 µL respectively, is used to aliquot optimized volumes of a reporter gene and a lipid-based transfection reagent (Roche’s Fugene 6) to each well and incubated for at least 15 minutes at room temperature in order for the spotted cDNA, reporter cDNA and the transfection reagent to complex. During this incubation period, cells can be prepared. Also using the Titertek 384 MultiDrop, resuspended cells are aliquoted over the DNA:lipid complex and incubated for overexpression for 24-48 hours. 24-48 hours post-transfection, the plates can be read using FLIPR, LJL Biosystems Acquest (Luminescence/Fluorescence), or Q3DM Imaging Microscope (phenotypic screen). Ex/ For luminescence readout : After the 48 hour incubation, the MultiDrop is used to dispense Promega’s Bright-Glo Luciferase Assay System, a system used to quantify the amount of firefly luciferase expression, over the cell:lipid:DNA mix, incubated for approximately 2 minutes and then read on either the Acquest or Q3DM Imaging Microscope. LJL Biosystem’s Acquest measures both fluorescence and luminescence readouts. After readout, the raw data is then submitted for statistical analysis. For optimization purposes, three smaller scale screens must be run (0.3K-1K), using the same process described above, before moving on to the larger screen.