Parallel analysis of v-Src mutant protein function using reverse transfection cell arrays.

The conversion of the genomic information produced by the recent sequencing projects into a comprehensive understanding of the human proteome has yet to occur. A new technology that represents a potential bridge between genomics and proteomics is reverse transfection. Reverse transfection cell microarrays are produced by overlaying cDNA arrays with mammalian cells, generating localized clusters of transfected cells with each cluster overexpressing a unique protein. This miniaturized cell-based microarray format affords parallel functional analysis of thousands of cDNA constructs in a high throughput format. In this report we document the development of a co-transfection methodology for reverse transfection applications. The demonstrated high co-transfection efficiency with a "marker" plasmid encoding for GFP enables the identification of transfected cells and eliminates the need for epitope-tagged constructs in cell-based high throughput screening applications using reverse transfection. This co-transfection method was used to study in parallel the structure/function of multiple versions of the v-Src protein using automated fluorescence microscopy. The wild-type v-Src protein and four mutants having insertions or deletions in the SH2 or SH3 domains displayed high levels of tyrosine kinase activity in HEK293T cells. Three other mutated v-Src proteins, including a kinase-dead version, were shown to be defective for tyrosine kinase activity. This reverse co-transfection approach is applicable for high throughput screening of both cDNA libraries and positional scanning recombinant protein libraries.

In situ mechanical interferometry of matrigel films.

Many biological materials and cell substrates are very soft (Young's modulus <500 Pa) and it is difficult to characterize their mechanical properties. Here we report local elasticity of the surface layers of Matrigel films used for cell culture. We used a new measurement technology, mechanical imaging interferometry, to obtain point mechanical measurements over micron-sized areas. The median values of 650 +/- 400 Pa (# measurements, n = 50), determined by the Hertz contact model, agree well with bulk measurements; however, on the microscale, the films were heterogeneous and contained regions distinctly stiffer than average (1-2 kPa). The first measurement of yield strengths of 170 +/- 100 Pa (n = 43) indicates that Matrigel films deform plastically at stress levels of similar scale to cell tractional forces.