Targeted Gene Manipulation in the Mouse

Targeted gene expression has become a standard technique in Drosophila labs, but the same cannot be said for biologists working on gene expression in more complex organisms such as mammals. Targeted, conditional gene expression even in well-established mammalian model organisms, such as the mouse, is a technically challenging and costly endeavor for most labs. Recently, (Matsuda and Cepko) addressed this issue in elegant detail, demonstrating the ability to temporally and spatially control gene expression in the mouse retina and brain using combinations of in vivo electroporation, Cre/loxP-mediated inducible vectors, and cell-specific promoters. Initially, three inducible Cre expression vectors and a single FLP recombinase-based vector were in vivo electroporated and tested. The target vector contained LoxP sites flanking copies of inactive GFP or DsRED, which, upon recombination by Cre-recombinase, result in expression of GFP or DsRED as a reporter of activity. Following in vivo electroporation but prior to induction, three out of the four vectors showed GFP or DsRED fluorescence indicating “leakiness” of expression from the Cre gene. One of the vectors, ER^T2CRE^T2, showed no fluorescence prior to induction and was therefore used for further experiments. To examine spatial control of gene expression, the authors tested nine different retinal cell-specific promoters and were able to demonstrate, in most cases, tight spatial control of GFP and DsRED expression patterns. Moving a step further, combining the temporal control of the inducible Cre-recominbase vector ER^T2CRE^T2 with the spatial control of the retina-specific promoters, the authors demonstrated the ability to express reporter genes specifically in differentiated Müeller glial cells of the retina. Finally, to further illustrate the utility of these techniques, two additional experiments were described, demonstrating the ability to perform conditional RNAi and controlled misexpression of transcription factors in a variety of retinal cell types. The work presented here demonstrates that temporally and spatially controlled gene expression can be efficiently accomplished in the mouse using in vivo electroporation technology. This work should open new doors for studying in vivo gene expression and function in mammals.

-Matsuda and Cepko. 2007. Controlled expression of transgenes introduced by in vivo electroporation. Proceedings of the National Academy of Sciences of the USA 104(3):1027-1032.

Predicting Alternative Promoters

Promoters are DNA sequences that act as regulators of gene expression. It has recently been reported that a significant number of human genes are potentially under the regulatory control of alternative promoters. The biological and regulatory roles of alternative promoters (APs) are not well understood, making predictions about APs from sequence data very difficult. (Baek et al). attempt to shed light on APs by characterizing the difference between APs and single promoters (SPs), then using this information to design a discriminator to identify APs and SPs from gene sequence data. The authors used 12,025 promoter regions conserved between mouse and human for the analyses. From the data set, 1018 APs could be confidently assigned, while 3109 SPs were identified. Sequence analysis demonstrated that APs had higher sequence conservation when compared to SPs, with CpG island-rich APs being the most highly conserved. The authors also searched APs and SPs for transcription factor binding sites, finding that APs were overrepresented by hexamers associated with tissue-specific transcriptional activators and context-dependent activators/repressors. Biological function relationships for APs and SPs were also examined, showing that SPs were frequently associated with housekeeping genes while APs were often found with genes involved in transcriptional regulation and development. Utilizing these observations, an approximate log likelihood ratio discriminator was formulated for predicting APs and SPs. In a test of the new discriminator, 74% of the predicted SPs showed experimental evidence of a single promoter, while 64% of APs identified showed evidence of multiple promoters for an overall accuracy rate of 69%. This work provides not only an overview of APs compared to SPs in the mouse and human genomes, but also a predictive discriminator to make identification of APs easier in the future.

-Baek et al. 2007. Characterization and predictive discovery of evolutionary conserved mammalian alternative promoters. Genome Research 17(2):145-155.

So Many Spectra, So Little Time

Tandem mass spectrometry (MS/MS) in combination with liquid chromatography is a powerful method to identify proteins in a high-throughput manner. As instrument accuracy increases, computer algorithms to rapidly and reliably identify each peptide spectra and determine the full protein identity are becoming increasingly critical. Most current methods to identify peptide spectra rely on comparisons between the experimental spectra and databases of theoretical spectra predicted from protein databases. However, this method is computationally demanding when data from high-throughput proteomic studies need to be analyzed. (Liu et al). examined a computationally more efficient method that compares experimental spectra to a library of previously identified peptide spectra. The value of this method, over the widely used current method, is that this new method takes into account instrument-dependent factors and peptide-specific differences when looking at fragmentation possibilities. Two data sets consisting of 7189 unique peptide spectra were used for the computational experiments. The authors found that peptide spectral peak intensities tended to follow Poisson distributions, which allowed the data to be transformed using a square-root transformation, producing more stable peak variation. This simple transformation greatly improved the accuracy of the spectral matching. To compare the spectral similarities, the authors found that correlation coefficients were the most robust measure. Finally, when assembling multiple spectra for the same reference peptide, a simple average demonstrated the best overall accuracy. Although previous work has utilized spectral comparisons for MS/MS data, here the authors have provided analyses demonstrating new computational approaches to rapidly and accurately identify peptides and proteins following shotgun MS/MS experiments. These computational methods will surely be used in the future, improving the ability to identify proteins from large-scale proteomic experiments.

-Liu et al. 2007. Methods for peptide identification by spectral comparison. Journal of Proteome Research 5:3.

Visualizing Enzyme-Substrate Complexes

The ability to visualize enzyme-substrate interactions in live cells could reveal much information regarding enzyme regulation and activity. (Yudushkin et al). have demonstrated that, using a method based on Förster resonant energy transfer (FRET), imaging of enzyme-substrate complexes in live cells can be accomplished. In the work, proteintyrosine phosphatase PTP1B, which acts to dephosphorylate receptor tyrosine kinases to terminate growth factor signaling, was examined using the new FRET-based method to better understand PTP1B activity in cells. To demonstrate the feasibility of studying enzyme-substrate complexes in live cells, a PTP1B variant lacking the catalytic activity but retaining substrate binding ability was fused to a genetically encoded fluorescent protein as the donor chromophore, while a conjugated substrate, a synthetic phosophotyrosine-containing peptide, acted as the acceptor chromophore. Interactions between the donor and acceptor were visualized and quantitated using fluorescence lifetime imaging. These experiments showed the interaction between PTP1B and substrate could be visualized in cells. Wild-type PTP1B was next analyzed for interaction with the synthetic substrate in COS7 cells, which express the epidermal growth factor receptor endogenously. The results showed an initial uniform distribution of enzyme-substrate complexes in the cell followed by a gradient of enzyme-substrate complexes after one minute. Further experiments demonstrated that the enzyme-substrate complexes were maintained through phosphorylation-dephosphorylation cycles. Building on these results, the authors examined the exact spatial distribution of enzyme-substrate complexes throughout the cell and found that spatial regulation plays an important role in PTP1B activity and signaling. This elegant piece of work clearly shows the usefulness of the new FRET-based method to study enzyme-substrate intermediates and suggests this technique can be used to study any enzyme and substrate combination in live cells.

-Yudushkin et al. 2007. Live-cell imaging of enzyme-substrate interaction reveals spatial regulation of PTP1B. Science 315(5808):115-119.