The authors show that the combination of genetic elements in an expression cassette influences FP expression up to threefold, and that relative expressed brightness of individual FPs varied up to tenfold in relation to one another.
To quantify the effects of intrinsic and extrinsic variables that affect both the expression and brightness of FPs, we generated a loxed cassette acceptor (LCA) allele for the ubiquitously expressed Gt ROSA)26Sor (ROSA26) gene (Zambrowicz et al., 1997; Kohlhepp et al., 2001; Safran et al., 2003).
For normalization of expression data, the average brightness of the Cy3 and Cy5 channels respectively was used that was calculated from spots within a range from 30%to85%5% of the signal intensity distribution of all spots.
After 2 hr of expression, the sizes and brightness of cavin1-GFP assemblies were very similar to the ones obtained for MCF-7 cells, indicating that we could use the cell-free system to reproduce the behaviour of cavin oligomerization.
As is well known, a number of factors influence apparent brightness of an FP beyond protein expression, including the efficiency and rate of maturation, the molar extinction coefficient values within the excitation wavelength range, and the quantum yield (Olenych et al., 2007; Rizzo et al., 2009).
The scaling of a spectrum in the visible range is in general an expression for the brightness of the sample, which means some fish among the samples set appear brighter than other.
To quantify the factors that determine both the expression and relative brightness of FPs in mouse embryonic stem cells (mESCs) and in mice, we generated eight different FP-expressing ROSA26 alleles using recombinase-mediated cassette exchange (RMCE).
The distribution of expression within a probeset, leading to a detection p-value, is influenced by all stages of the microarray process including scanner brightness, background, RNA quality, chip design, etc. [ 5].
Utilizing the ROSA26 LCA allele, we generated eight different FP reporter alleles by RMCE, which were used to assess the effects of several regulatory elements on FP expression and to compare the brightness of five different FP variants.
The scaling factor is inversely related to the overall brightness of the chip and provides a measure of the overall expression level of the array and was again, comparable for all 3 labeling approaches (Table 1).
