The classical method for gene modification is homologous recombination. The benefit of recombination is its use of recombinases, which allow DNA segments to become inverted or deleted (e.g. to ‘knock-out’ a gene). They can also start translation at designated sites. This ability to accurately pinpoint genes is what initially allowed researchers an extra degree of freedom to the otherwise riskier approach of gene-knockout.
Today, recombinases provide researchers with an ability to precisely control the gene activity in vivo. This approach has been the widely preferred method to generate a germ line ‘knock-out’ or ‘knock-in’ within mouse embryonic stem cells [1]. One disadvantage to using the classical approach is that it typically takes more than a year to generate a genetically modified mouse. These kind of applications, however, have added greatly to our understanding of gene activity in vivo. The use of recombination continues to deliver fundamental insights into the most complex biological phenomena such as development, behavior, and disease.
The classical method for gene modification is homologous recombination. The benefit of recombination is its use of recombinases, which allow DNA segments to become inverted or deleted (e.g. to knock-out a gene). They can also start translation at designated sites. This ability to accurately pinpoint genes is what initially allowed researchers an extra degree of freedom to the otherwise riskier approach of gene-knockout.
Today, recombinases provide researchers with an ability to precisely control the gene activity
in vivo. This approach has been the widely preferred method to generate a germ line knock-out or knock-in within mouse embryonic stem cells [1]. One disadvantage to using the classical approach is that it typically takes more than a year to generate a genetically modified mouse. These kind of applications, however, have added greatly to our understanding of gene activity in vivo. The use of recombination continues to deliver fundamental insights into the most complex biological phenomena such as development, behavior, and disease.
The use of recombinases also revealed that many genes are active only within a distinct time window. And for those genes to function properly also depends on the rate of expression, which can be influenced by outside environmental signals [2].
Experiments with recombinases have shown that misexpression of a gene (e.g., either over- or under-expression of a gene, or expression outside the normal developmental programming) results in pathologies [3]. Mimicking this misexpression in transgenic mice has led to formation of numerous disease models that are of particular interest whenever the disease-causing gene activity can be controlled from outside.
Principles of recombination
Genetic engineering can be defined as the use of recombinant DNA and other biological and bioengineering techniques for directed modification of cellular processes and properties through either the introduction, deletion, and/or modification of genetic material to enhance function or production of normal physiological processes. These can occur through two means, either 1) gene knockouts and 2) gene overexpression.
Presently, the site-specific recombinases Cre and Flp are most frequently used for regulating gene activities [4]. They target a very specific 34-bp landing site, loxP and FRT, respectively, and begin recombination at these sites (see figure 1).
Similar attempts at using homologous recombination in human cells have appeared to be extremely challenging, since many diseases and cancers are manifested not through the expression of certain genes, but through the over-translation of a particular gene (or sets of genes).
Figure 1. Controlling gene expression with DNA recombination
Alternative approaches have emerged in their place in order to knock down gene expression, such as antisense oligonucleotides and short interfering RNAs (siRNAs), both have instead become the standard.
At a recent meeting hosted by the Association for Research in Vision and Ophthalmology (ARVO), researchers there described new cutting-edge technology, such as lipid-mediated siRNA knockdown of mRNA, using custom-built nanomaterials for gene delivery, and engineering stem cell sheets for corneal transplants. Presenter Alfred Lewin, Ph.D., professor of molecular genetics and microbiology at the University of Florida College of Medicine said, These tiny RNA molecules do not encode proteins or stable RNAs such as ribosomal RNA or transfer RNA. Rather, they influence the transcription and translation of messenger RNA.
So while these approaches can be effective at transiently reducing gene expression, the effects are usually incomplete, short-lived, and can often affect off-target genes [5]. These shortcomings have fueled the demand for more effective methods of gene modification.
A new wave of technology that is variously termed "gene editing," "genome editing," or "genome engineering" emerged as a way to address this demand by giving investigators the ability to accurately pinpoint a variety of genetic alterations in mammalian cells, ranging from knock-in/knock-out of single nucleotide variants to insertion of whole genes to deletion of chromosomal regions.
This past decade we have witnessed rapid innovations in gene-editing technology. The opportunity that exists right now for researchers and investigators is the additional ability to manipulate virtually any gene within a variety of cells and organisms.
DNA sequencing technologies have revolutionized the field of microbiology, spawned a new field called metagenetics, and has created a deluge of previously unknown data. This rapid development in genomics is generating a highly efficient, precise, and continually more cost-effective means to refine advanced models of disease in both human and animals.
Over the last few blogs, we have described the key advantages and disadvantages of the four most popular genome-editing tools. These descriptions are not meant to be a comprehensive review of the work leading to the development of the tools, but rather to give readers a working knowledge of the tools available and the ability to select among them for desired tasks.
Next Generation Sequencing
A quick word on Next Generation Sequencing (NGS). All genetic studies, to date, utilize three main NGS platforms: capillary-based sequencing (Sanger, such as Applied Biosystems 3730xl), pyro-sequencing (including Roche 454 GS, FLX, and FLX Titanium), and Illumina clonal arrays (Illumina GAIIx, HiSeq 2000, and MiSeq). Using each technology has distinct advantages, such as read length, depth of coverage, accuracy, scalability, cost, as well as time to generate the data.
Unlike molecular genetics, there is no standard sequencing platform, methodology, or computational tool used by scientific researchers, making any sort of cross-comparison difficult, at best. The choices researchers make for which technology or bioinformatics tools to use are critically important considerations for the experimental design and interpretation of study results.
SOURCES
[1] Grindley ND, Whiteson KL, Rice PA: Mechanisms of site-specific recombination.
Annu Rev Biochem 2006, 75: 567-605.
[2] Espeli O, Marians KJ: Untangling intracellular DNA topology. Mol Microbiol
2004, 52: 925-931.
[3] Sherratt DJ, Soballe B, Barre FX, Filipe S, Lau I, Massey T, Yates J:
Recombination and chromosome segregation. Philos Trans R Soc Lond B Biol
Sci 2004, 359: 61-69.
[4,5] Hallet B, Vanhoof V, Cornet F: DNA site-specific resolution systems.
Plasmid Biology Edited by: Funnell B, Phillips G; Washington, DC: ASM Press;
2004: 145-179.