Bio154JM08/Toolbox/Paper 3

FLP/FRT system
FLP/FRT Recombination is a directed recombination technology involving the recombination of sequences between FRT sites by the flippase recombination enzyme derived from Saccharomyces cerevisiae. This allows the precise manipulation of an organism's DNA under controlled conditions in vivo.

FLP/FRT is a technique for inducing mosaics of +/+ and -/- (mutant, or carrying an allele of interest) cells in heterozygote organisms. This technique is particularly useful when dealing with traits that are necessary for development in the entire organism thus preventing the use of a homozygous organism. The FLP/FRT system enhances the rate of mitotic recombination, which refers to crossing over in mitosis rather than meiosis, and is ordinarily very rare. By expressing an FRT region nearer to the centromere than the gene of interest, the chromosomes may cross over at this point, so that each chromosome will have a chromatid with the + allele and one with the - allele, instead of having identical chromatids as usual in mitosis. The two chromosomes will separate independently in mitosis, so there is a 50% probability that one daughter cell will have both - alleles and the other will have both + alleles. The crossing over at the FRT sites is mediated by FLP, flippase recombination enzyme derived from Saccharomyces cerevisiae. Depending on the experimental design, it may be expressed in a heat-inducible form or regulated by a tissue-specific enhancer.

MARCM system
In Mosaic Analysis, it is sometimes inconvenient to use negatively marked clones, especially when generating very small patches of cells, where it is more difficult to see a dark spot on a bright background than vice versa. It is possible to create positively marked clones using the so called MARCM system, which stands for "Mosaic Analysis with a Repressible Cell Marker". This technique was developed by Liqun Luo, a professor at Stanford University. In this system the GAL4/UAS system is used to globally express GFP. However, the gene GAL80 is used to repress the action of GAL4, preventing the expression of GFP. Instead of using GFP to mark the wild type chromosome, GAL80 serves this purpose, so that when it is removed, GAL4 is allowed to function, and GFP turns on. This results in the cells of interest being marked brightly in a dark background.

GAL4-UAS system
The GAL4-UAS system allows for targeted gene expression in vertebrates using the transcription factor GAL4 and the gene of interest attached to a promoter specific to GAL4, the Upstream Activating Sequence (UAS). The system was originally discovered in the yeast Saccharomyces cerevisiae.

The system works by creating two transgenic strains of the organism and crossing them to yield progeny with both transgenes. In one strain, an activator line is introduced by inserting the gene for GAL4 near a specific promoter with a known expression pattern (e.g., a promoter used in the development of Drosophila melanogaster legs), while, in the other strain, an effector line is created by fusing the UAS upstream of the gene of interest. When the two lines are crossed, the progeny with both transgenes will express the gene of interest according to the expression profile of the promoter near which the GAL4 gene was placed. It is by this control over the insertion of the GAL4 gene that targeted expression (e.g., ectopic expression or expression of mutated genes) can be achieved.

Heat shock
Heat shock proteins (HSP) are a part of the cell's internal repair mechanism. They are also called stress-proteins. They respond to heat, cold and oxygen deprivation by activating several cascade pathways. applying a heat shock means subjecting cells to a higher temperature than the ideal body temperature of the organism from which the cell line was derived. Heat shock is a method in which genes can be introduced into a vector.

Confocal imaging
Confocal imaging is a microscopy technique used to obtain high resolution images and 3-D reconstructions.

In confocal microscopy, one or more beams of light are focused by an objective lens on to a small volume of a fluorescent specimen. The fluorescent light emitted from this small area is separated from the source laser light by a beam splitter. The fluorescence is detected by computer detection device. This process to obtain the information for a single pixel is repeated many times as the laser is scanned over a single plane. After scanning several layers of a specimen, the computer reconstructs a 3-D image by stacking the 2-D planes together.

Confocal imaging has several advantages. Confocal microscopy has better resolution than other microscopy techniques. It also allows for images of thick, living specimens to be obtained with minimal preparation. Note: Fluorescence is required for this technique. This can be achieved by using fluorescent dyes or by transgenically expressing fluorescent proteins such as GFP.

For some sample images, click here.

Frozen sectioning
In Discussion Paper 3 (Wernet et al, 2006), mid-pupal and adult cryosectioned (frozen sectioned) retinas were immuno-stained for the various rhodopsin types. Frozen sections are often used in immunohistochemistry in order to preserve certain cell antigens that do not survive routine tissue fixation and/or paraffin embedding. However, the disadvantages of frozen sections include: poor morphology, poor resolution at higher magnifications, special storage requirements, limited retrospective studies, and increased cutting difficulty over paraffin sections.

There are two primary methods to sectioning frozen brains. The microtome is used for sectioning frozen, fixed brains. For this procedure, the fixed brains must be cryoprotected by infiltration of sucrose to prevent freezing artifacts during sectioning. Sucrose-infiltrated brains are frozen and maintained frozen during sectioning. Sections are collected off the microtome knife and placed in a buffered saline solution during sectioning.

Wernet et al. used 10 micrometer horizontal eye sections, sectioned using a cryostat. A cryostat is a microtome housed within a freezing chamber that allows the sectioning process to be performed at a temperature of -20 to -30 degrees Celsius. The cryostat is required for sectioning fresh-frozen brains, as the unfixed brain sections must be maintained in a frozen state until they are affixed to a microscope slide; however, cryostat sectioning may also be used for perfusion-fixed brains. Cryostat sections retain morphological integrity after being sections and can be affixed directly onto microscopic slides for immunohistochemistry use.

Water immersion microscopy
In this modified form of microscopy, water is placed between the front lens element and the specimen so as to increase the numerical aperture of the microscope system. Water immersion objectives allow for high-resolution imaging through aqueous layers on the order of 200-micrometers thickness. As with oil immersion microscopy, the main advantage of water immersion objectives is improved imaging capabilities in thick sections of biological specimens. However, water has distinct advantages over oil. It has no inherent fluorescence to complicate image interpretation, it’s very cheap, there is almost no risk of contaminating the specimen, and there is no special cleanup method required.