Seeing and Timing Gene Delivery

Simple, rapid methods for in vivo gene delivery have the potential to speed the process of scientific discovery. In this issue of BioTechniques, two new methods for in vivo gene delivery in the mouse are described. Current methods for gene delivery to the mammalian eye rely heavily on the use of viral carriers to mediate delivery. Virus-based methods can be extremely time-consuming, labor-intensive, and do not work for all neuronal cell types. Liao and Yau propose utilizing a different approach, employing the cationic polymer polyethylenimine (PEI) fused with a DNA of interest as the delivery vehicle. To explore this possibility, the authors delivered an shRNA-expressing plasmid, targeting the melanopsin gene, with PEI directly to retinal ganglion cells (RGCs) by intravitreal injection. The authors found that expression was confined to RGCs and resulted in a dramatic reduction in levels of melanopsin. To confirm a phenotypic change from the decreased melanopsin expression, mice were tested for pupillary reaction to flashes of bright light. Animals with decreased levels of melanopsin demonstrated altered pupil reaction when compared to controls, in agreement with previous work on pupil response to reduced melanopsin levels. Taken together, these results demonstrated effective reduction of melanopsin expression specifically in RGCs, in vivo, using PEI/DNA complexes. Gene delivery can rely on simple injection methods, as demonstrated by Liao and Yau, or sometimes simply by the correct timing. Georgiades et al. demonstrate that the proper timing of lentivirus infection can also allow efficient manipulation of gene expression, this time in the mammalian trophoblast. Developmentally, the trophoblast functions in the growth and development of the fetus, but does not give rise to the fetus. Exploring trophoblast-specific gene expression has been difficult due to the contributions of other cell types associated with fetal development. The authors present a simple, rapid method to manipulate gene expression specifically in the trophoblast. Using lentivirus vectors containing the green fluorescent protein (GFP) transgene added to isolated mouse blastocytes in cultured media, GFP expression was found to be restricted to trophoblast cell lineages. To demonstrate the potential utility of this new method, two additional experiments were described demonstrating the ability to silence genes in trophoblast-specific cells using shRNAs and knockout genes in these cell types utilizing the Cre-recombinase system. These two new gene delivery methods will help neuroscientists and developmental biologists to better understand gene function. -Pages 285 and 317

Bear Bones

Successful extraction of DNA from ancient samples requires achieving a balance between maximizing the yield of intact DNA and minimizing co-extraction of contaminating substances which may inhibit the downstream PCR. While published protocols and commercial kits for the extraction of ancient DNA abound, Rohland and Hofreiter present the first robust cross-comparison of existing methods for ancient DNA extraction; the authors applied these methods to Pleistocene cave bear bones and teeth and optimized those that produced the highest yields in a cost- and labor-effective manner, as assessed by quantitative PCR. After evauating a wide variety of parameters during the development of the optimized procedure, the authors provide a strong “minimalist” working protocol and detailed data to support their approach. While this protocol will be applicable to samples similar to those used in this study, this work will also serve as a model for similar studies with ancient samples of different tissue type and preservation conditions. -Page 343

The Powerhouse of the Cell Takes on the DNA Chip

Microarray analysis has proved a powerful method to study gene expression changes at a global level. One problem often encountered by researchers using microarrays is the best way to understand and interpret the mountains of data produced from these analyses. Constructing micro-arrays that are more specific is one way to decrease experimental noise and obtain biologically relevant data. Bai et al. provide a new microarray to specifically study gene expression changes for mitochondrial and mitochondrial-associated genes. The new chip, called hMitChip3, consists of an array of the 37 mitochondrial genes, 1098 nuclear-encoded mitochondrial related genes, and 225 controls. The hMitChip3 was demonstrated to have low background upon hybridization and a 13-fold greater signal-to-noise ratio. To experimentally test the new array, the authors compared a human melanoma cell line, which was rapidly dividing, to a slower dividing cell line. In total, 66 genes were found to be differentially expressed, including a number of genes involved in the process of cell proliferation. Six of the identified genes were validated for expression changes by quantitative RT-PCR, demonstrating the accuracy of the hMitChip3. To further complement the new hMitChip3, a bioinformatics database was constructed which included the annotation of 1135 of the test genes by categories such as molecular function, biological processes, cellular components, biochemical pathways, and genetic disorders. Taken together, the new hMitChip3 and the associated bioinformatics database will greatly aide researchers in the understanding human mitochondrial function. -Page 365

Validating a Multiplex ELISA

The capacity to identify multiple proteins simultaneously in a complex sample can significantly accelerate research efforts and can also aid diagnosis of diseases characterized by specific gene expression profiles. While a number of multiplex assays for this purpose have been described, rigorous comparisons of the multiplex assays to assays measuring the same antigens individually have not been reported. In this issue, Liew et al. compare the performance of their multiplex sandwich ELISA array (microELISA) in the identification and quantitation of nine antigens in clinical samples to the same analysis carried out in individual chemiluminescence-based immunoassays of the same antigens. In the microELISA antibodies directed against α-fetoprotein (AFP), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), cancer antigen 125 (CA 125), CA 15-3, CA 19-9, β-human chorionic gonadotrophin (β-hCG), luteinizing hormone (LH), and follicle stimulating hormone (FSH) were arrayed on the bottom of a single well of 96-well plate and exposed to aliquots of the same samples of human serum or plasma in which the antigens had been previously assayed individually. Deming regression analysis indicated that when identical reagents were used, there was no statistically significant difference in the results of the micro- and macroassays in 63% of the comparisons; this is a higher value than was observed when different antibodies were used in the two types of assays. The authors point out that continued development of databases of antibody reactivity will allow improved antibody selection and higher correlation values in this type of comparison. -Page 327

Dual Reporter Assay

Luciferase is widely used as a reporter due to its sensitivity and linear range, and a wide variety of luciferase-based systems are available for monitoring promoter activity in prokaryotic and eukaryotic cells. While these systems offer high sensitivity in diverse combinations of luciferases, many are unsuitable for use in living cells because cell lysis is required prior to measurement of bioluminescence produced by the action of the luciferase on its substrate. Wu et al. have devised a reporter system that incorporates two secreted luciferases, one produced by the marine ostracod crustacean Cypridina (CLuc) and the second by the marine copepod Gaussia (GLuc). Luciferase activity can be assayed in the medium of cultured cells, circumventing the need for cell lysis and permitting monitoring of promoter activity in a single population of cells over hours or days. Because GLuc activity is inhibited by SDS, the assay can be performed in a single tube, assaying GLuc activity, followed by addition of SDS, and measurement of CLuc activity. -Page 290