Brainwashing

Primary cells, such as T cells and neurons, can be extremely difficult to transfect, often requiring the use of time-consuming and sometimes costly viral-based methods. Buchser et al. have developed an inexpensive means of simultaneously performing multiple transfections using electroporation, varying only the gene to be electroporated from well to well in a 96-well plate. The authors used cerebellar granule neurons isolated from 8- to 10-day-old wild-type mice and were able to electroporate a mere 25,000 cells per transfection well. This is significant, as primary cells are limited in number; most other transfection strategies require at least 10⁶ cells. They achieved transfection efficiencies of 26.8%, with GFP expression levels as high as 100 standard deviations above nontransfected levels. Those cells that survived the procedure remained healthy and viable after transfection. To date, the lack of effective methods for expressing cDNAs in mammalian neurons has limited the utility of high content screening (HCS) in these cells; this electroporation approach should enable HCS genomics studies and examination of how various agents in drug and compound libraries affect neuronal growth, differentiation, and morphology. -Page 619

Flash Point

A new class of fluorescein-related protein labeling reagents has recently been developed based on the interaction between FlAsHs-EDT₂ (a nonfluorescent biarsenical derivative of fluorescin, bis-ethanediol adduct) with the amino acid sequence Cys-Cys-Xaa-Xaa-Cys-Cys (the tetracysteine, or TC, motif). Four covalent bonds are formed between the two arsenic groups in the FlAsH and the two thiol groups in the TC-tag. Two major advantages of this technology over previous methods are the relatively small size of the TC-tag and the ability to pulse-chase the fluorophore. Estévez and Somerville have applied FlAsH to plant cell biology, with impressive results. Using both tobacco and Arabidopsis leaves, they demonstrate that the expression and localization of several synthetic glycomodule peptides can be followed in live cells. They also used ReAsH-EDT₂, a derivative of the red fluorophore resorufin, although it had a higher signal-to-noise ratio than FlAsHs-EDT₂. -Page 569

It's a Dirty Assay, but Somebody's Got to Do It

Drug resistance, whether innate or acquired over the course of treatment, is one of the banes of chemotherapy. In order to better understand the mechanisms underlying drug resistance, cell survival after different treatments must be determined. Dictyostelium discoideum, an amoeba that lives in soil, is increasingly being used as a primary system in these cell survival studies. However, cell viability in D. discoideum has traditionally been measured by counting plaques. This method is labor-intensive, subjective, and slow. Now, Min et al. have adapted a commercially available luciferase-based assay to determine cell viability in populations of D. discoideum cells. The approach works by measuring the amount of ATP in metabolically active cells. The authors demonstrate the effectiveness of their assay by treating the cells with cisplatin, DMS, and 4NQO. Each of these drugs works differently—cisplatin forms intrastrand GpG adducts within DNA, preventing mitosis; DMS inhibits sphingosine kinase; and 4NQO is a γ irradiation mimetic. In all cases they were able to reproduce previously published results. They suggest that this modified assay can also be used for drug screens and for screening mutant libraries. -Page 591

WGA ChIP-chip Beats LM-PCR ChIP-chip

With the chromatin immunoprecipitation (ChIP)-chip assay, ChIP has evolved from looking at one gene at a time to being more a global analysis tool. One ChIP sample does not yield enough DNA for labeling and hybridization to an array; therefore the DNA must be amplified. This amplification is usually achieved by ligation-mediated PCR (LM-PCR). O'Geen et al. have demonstrated that LM-PCR can produce very high background when samples are analyzed on genomic tiling arrays. They used ChIP samples amplified by LM-PCR to identify binding sites for E2F family members, but were unable to identify Oct4 binding sites this way, despite the confirmed presence of Oct4 sites in the array. They therefore compared methods for ChIP-chip and found that whole genome amplification (WGA) gave the best signal-to-noise ratios. This method was originally developed to provide accurate representation of the genome for SNP analyses in tumor samples and copy number change studies. In it, chromatin from 1×10⁷ cells is sonicated to generate fragments with an average size of 0.5–1 kb. This library is then primed so the fragments have defined 3′ and 5′ termini and replicated using first linear and then geometric amplification. -Page 577

A Bone to Pick

A multiplex high-throughput assay for osteogenesis could analyze the effects of environmental factors on osteoblast differentiation and could screen different bone marrow samples for their osteogenic capabilities. Most studies of osteoblast differentiation have used either primary cultures of osteoblasts expanded from rodent calvarial collagenase digests or bone marrow stromal cells flushed from mouse long bones, but the selective culturing times and conditions inherent in these methods can affect the outcome of drug studies. To circumvent this obstacle, Sharma et al. used unexpanded bone marrow-derived osteoblasts. They have developed a 96-well alkaline phosphatase-based high-throughput assay that can be combined with multiparameter analysis for studying the various processes and factors involved in osteoblast differentiation. An alkaline phosphatase substrate that produces a yellow colored product, p-nitrophenyl phosphate (pNPP), is added to the cells, and absorbance is measured at 405 nm. Since alkaline phosphatase expression is highly elevated in differentiated osteoblasts, this absorbance correlates nicely with osteoblastic colony forming units. The same plate, once rinsed, can then be used for the detection of additional markers using standard staining and immunohistochemical techniques. This sequential assay design also works well with homogenous murine osteoblast cell lines. -Page 539

Don't SNP This Worm

SNPs occur frequently in the individual genomes of heterogeneous populations. Thus, SNP mapping is quickly becoming the preferred method for performing linkage studies and analyzing genetic variation. However, many of the quantitative methods that have been developed for high-throughput SNP analysis are both expensive and complicated. Shelton has created a simple method for linkage analysis and SNP detection in Caenorhabditis elegans using unmodified oligonucleotides in a standard quantitative PCR detection platform. He demonstrates that this method is capable of detecting specific SNP variations between N2, the laboratory wild-type strain of C. elegans, and CB4856, a polymorphic isolate from Hawaii that is commonly used in mapping studies. This method should be of interest to researchers in a number of fields—those designing and using large numbers of SNP assays for linkage mapping and gene association studies in different species will appreciate the description of automated oligonucleotide design for the quantitative PCR assay using PERL-based programming scripts; and those in the C. elegans community will appreciate the 923 SNP-specific oligonucleotide pairs he designed. -Page 583