LSI Issue 25

Taregeted Genome Editing Download
Targeted Genome Editing Using Engineering Zinc Finger Nucleases
Phil Simmons and Derek Douglas
Product Management, Biotechnology Division, Sigma-Aldrich
The determination of complete genome sequences for a wide variety of experimental organisms established a first step in the search for an in-depth understanding of the complex genetic functions that define living entities. Extensive genetic, biochemical, cytological and physiological analyses are now required to correlate genome sequence with the next level of understanding of genetic function. As such, technologies that allow researchers to routinely and efficiently edit the genomes of virtually any species, by directing mutations in a truly targeted fashion, would greatly enhance the understanding of basic biology, and potentially lead to novel ways of treating human disease.
Early Approaches to Targeted Genome Editing
While early approaches to genetic manipulation used random and/or non-targeted methods such as ionizing radiation and chemical-induced mutagenesis to make changes to the genome, more recent methods have employed targeted methods of genomic editing. The most well studied methods have relied on the process of homologous recombination (HR), a naturally occurring DNA-repair mechanism found in most cells that uses the second copy of a chromosome as a template to repair damage to a gene.
Starting with Nobel Prize winner Mario Capecchi’s earliest work, it was discovered that the HR process could be harnessed and directed in mouse embryonic stem cells (MESCs)... Read more

Dear Researcher,

Since the discovery of the double-helix structure of DNA by Watson and Crick some 60 years ago, we have seen many milestones that have irreversibly changed the path of life science forever. The discovery of the Polymerase Chain Reaction and the advancement of automatic sequencing protocols enabled breakthrough discoveries as to the identity of the genetic code that makes life possible.

This mapping of the genetic code, while a watershed moment, was only the beginning of the search for complete biological knowledge. The next act involved assigning function to the genetic code through technologies such as RNAi, a technology that for the first time gave researchers complete control over where and when genes were expressed.

As Sigma continues to be at the forefront of both the sequencing and RNAi revolutions, we are very excited to lead the next frontier of genomic research: the ability to precisely and permanently ‘edit’ the genome as easily as a writer would modify a letter or a poet compose a beautiful sonnet.

In this issue of Life Science Innovations, we will introduce you to CompoZr™ Zinc Finger Nuclease (ZFN) Technology for Targeted Genome Editing. With CompoZr ZFN Technology, the power to change the genetic code will be in your hands. Sigma will provide the technology and we ask you, our dear readers: “How will you use this technology in your research? How will the CompoZr ZFN Technology enable you to change the world?”

Good Luck and Good Research,

Enhancing Lentiviral Transduction Efficiency Download
Methods for Enhancing Lentiviral Tranduction Efficiency
Andrea Spencer
Research and Development, Biotechnology Division, Sigma-Aldrich
An experiment to directly compare three methods of lentiviral transduction of Jurkat cells was conducted in order to determine the method that yields the greatest transduction efficiency. Spinoculation was carried out in parallel with an overnight incubation of virus with cells in the presence of polybrene (hexadimethrine bromide), and with transductions conducted on fibronection-coated plates. Spinoculation was shown to be the most successful method of transduction for Jurkat cells.
RNAi is a useful tool for functional analysis of genes and developing a potential therapeutic strategy for various diseases. Unlike murinebased MMLV or MSCV retroviral systems, lentiviral-based particles permit efficient transduction and integration of a specific shRNA construct into differentiated and non-dividing cells.1 While incubation of lentivirus pseudotyped with G glycoprotein from vesicular stomatitis virus (VSV-G) and cells in the presence of polybrene can efficiently serve as the method of transduction for many cell types, some cells are more difficult to transduce. Therefore, modifications have been made to transduction protocols for hardto- transduce lines, such as T-cells, to facilitate binding of viral envelope protein to cells. Some of these modifications include... Read more
siRNA Design, Quality, and Delivery Download
Key Drivers of Performance in the siRNA Workflow
Steven Suchyta
Product Management, Biotechnology Division, Sigma-Aldrich
Overview of siRNAs and RNA Interference
RNA interference (RNAi) is a natural biological mechanism wherein short inhibitory RNA (siRNA) duplexes induce potent inhibition of gene expression. These siRNA duplexes are produced naturally when an enzyme, Dicer, cleaves long double-stranded RNA (dsRNA) into smaller fragments. The resulting 21-23 nucleotide dsRNA fragments, termed siRNAs, then associate with an RNase-containing complex to form the RNA-induced silencing complex (RISC). The RISC unwinds the duplex and releases the sense strand. The RISC-bound antisense strand then serves as a guide for targeting the activated complex to complementary mRNA sequences. This results in subsequent mRNA cleavage and degradation. In effect, only catalytic amounts of siRNA are required for destruction of mRNA, resulting in the knockdown or silencing of the target gene and diminished protein expression.
This elegant RNAi mechanism has been quickly adopted by the research community as a method for targeted gene expression knockdown. Gene expression silencing has become a very important strategy in functional genomics. Optimized siRNA reagent kits and protocols have now made RNAi experiments fast and convenient. More importantly, the availability of extensive siRNA libraries, high-throughput screening (HTS) platforms, and bioinformatics software... Read more
TransPlex® RNA Amplification Kit Download
TransPlex Complete Whole Transcriptome Amplification Kit: Whole Transcriptome Amplification of Highly-Degraded RNA from FFPE Tissues
Ken Heuermann and Brian Ward
Research and Development, Biotechnology Division, Sigma-Aldrich
The TransPlex Complete Whole Transcriptome Amplification (WTA2) Kit effectively amplifies intact and highly degraded RNA. To benchmark maintenance of representative RNA levels during amplification, differential gene expression of human liver and brain tissues was examined by microarray analysis. Expression profiles of high-quality RNA amplified with the TransPlex Complete WTA2 Kit or Eberwine linear amplification were compared with that of unamplified cDNA. Results indicate that the TransPlex Complete WTA2 Kit and unamplified profiles correspond closely. Effective amplification of RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue was demonstrated by comparing the array results using target prepared from frozen or FFPE malignant prostate samples, versus matched frozen normal tissue. KEGG pathway profiles and global transcriptome analysis reveal that amplified FFPE target performed comparably to that of frozen malignant tissue. The TransPlex Complete WTA2 Kit is able to amplify nanogram quantities of intact total RNA or highly degraded RNA from FFPE tissue samples while maintaining transcript levels representative of that of the unamplified input RNA. Read more
Prestige Antibodies® Download
Monospecific Antibodies Designed for Immunohistochemical Analysis
Sara A. Gunner&areing;s, Charlotta Agaton, Soraya Djerbi and Marianne Hansson
Atlas Antibodies AB, AlbaNova University Center, SE-106 91 Stockholm, Sweden
Well-characterized antibodies are essential tools for protein studies, global proteomics analysis, as well as for clinical diagnostics. Although production of antibodies is a well-established process and a large number of antibodies are commercially available through many vendors, specific antibodies still do not exist for the majority of human proteins. An underlying factor limiting the available antibody repertoire is that commercial production tends to focus on popular targets. The current needs of the proteomics community demand a far more global approach. A second significant issue is the lack of a universally defined standard for antibody quality. This makes it difficult to compare antibodies of various sources without committing resources to using the antibody in its final application. Initial standardized testing for specificity and sensitivity followed by a thorough characterization would be of great interest to the end user, but this is a costly endeavor and most efficiently accomplished on a larger scale.
At present, there are only a handful of large-scale high-throughput antibody production efforts initiated around the world.3 One such initiative is the Swedish Human Protein Atlas (HPA) program.4 The aim of this program is to explore the entire human proteome using an antibody-based proteomics approach.5,6 Specifically, the HPA program generates protein expression profiles of the non-redundant set of human proteins, presented as immunohistological... Read more