Trends in Biotechnology 110512 3d Reporter Genes

Reporter Genes. A. Connected to the gene of interest and used to indicate if the desired DNA is expressed. B. Allows researchers to measure how well a gene is expressed.

Reporter Genes A reporter gene is connected to the gene of interest and used to indicate if the desired DNA is expressed. It allows researchers to measure how well a gene is expressed. Here are some examples:

Reporter Genes β-glucuronidase gene (GUS) - an enzyme that breaks down chemicals called β-D-glucuronides. Can produce a blue or fluorescent color.

Reporter Genes Luciferase gene—found in firefly and the bacteria Vibrio harveyi, the gene produces light in response to the molecules luciferin and ATP.

Fig. 3.19 A tobacco plant transformed with the firefly luciferase gene.

Reporter Gene Green fluorescent protein (GFP) -produced by the jellyfish Aequorea victoria and interacts with the protein aequorin to produce fluorescence. The GFP gene can be fused with another gene, allowing GFP to indicate the production of the desired protein.

The jellyfish Aequorea victoria showing the fluorescence, and a model of the protein.

The green fluorescent protein (GFP) can be expressed in mammals.

Baby monkeys which have been engineered to express GFP.

GFP has been mutated to produce different colors.

GFP has been engineered (amino acid substitutions etc) to produce many colors. It has also been used to engineer brain cells to show different expressions.

In this Harvard University photograph released October 8th, 2008, brain cells of a laboratory mouse are shown glowing with multicolor fluorescent proteins at Harvard University in Cambridge, Mass. The Nobel prize in chemistry was awarded to two Americans and a U.S.-based Japanese scientist for research on a glowing jellyfish protein that revolutionized the ability to study disease and normal development in living organisms. (AP Photo/Harvard University, Livett-Weissman-Sanes-Lichtman)

Video: Brainbow http://www.youtube.com/watch?v=y51la5gS5Ws

Southern Blot Hybridization. Another way to see whether a particular DNA is present was invented by Edward Southern in the mid-1970s.

How to do it (Figures 3.20): 1. DNA fragments in a gel are denatured by alkaline buffer. 2. DNA fragments are transferred to a nylon or nitrocellulose membrane. 3. DNA probes are hybridized to the membrane, and the membrane will be exposed to show a band representing where the probe successfully hybridized.

Fig. 3.20 The steps involved in conduction a Southern blot hybridization.

Southern blotting and hybridization with probes can be used to locate a few specific fragments in a large pool of DNA.

Sensitivity of Southern blots have been increased by: 1. Increased activity of labeled probes. 2. Development of nonradioactive (color, fluorescent, or light-producing) probes. 3. Treatments to prevent nonspecific binding of probes to membranes. 4. Use of detectors (phosphoimagers) to obtain images of hybridization signals. 5. Use of vacuum blotting or electroblotting to reduce the DNA transfer time. 6. New methods of DNA transfer, such as downward capillary transfer.

Fig. 3. 21 Southern blot capillary DNA transfer Fig. 3.21 Southern blot capillary DNA transfer. (a) Downward capillary transfer.

Fig. 3.21 (b) Bidirectional capillary transfer.

Southern blot hybridization can be used to detect banding pattern produced by restriction enzymes, called “restriction fragment length polymorphisms” (RFLPs).

Restriction Fragment Length Polymorphism Video: http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

Southern blot hybridization is different from colony hybridization in the following ways: 1. Blotted DNA can be cloned or not cloned, and does not have to be in vectors. 2. DNA is separated on an agarose gel and transferred to the membrane, and bacterial colonies do not need to be lysed to release DNA.

Northern Blot Hybridization. A. RNA is transferred from a gel to a membrane, and DNA probes are used. B. Used to measure the quantity and determine the size of transcribed RNAs.