Question

Assess the use genetic mutant analysis and reporter genes (green fluorescent protein) tools in the study of the of development. What are the advantages of these tools? Do they have any limitations or restrictions?

Answer 1

use of genetic mutant analysis

Genetic disorders are traditionally categorized into three main groups: single-gene, chromosomal, and multifactorial disorders. Single gene or Mendelian disorders result from errors in DNA sequence of a gene and include autosomal dominant (AD), autosomal recessive (AR), X-linked recessive (XR), X-linked dominant and Y-linked (holandric) disorders. Chromosomal disorders are due to chromosomal aberrations including numerical and structural damages. Molecular and cytogenetic techniques have been applied to identify genetic mutations leading to diseases. Accurate diagnosis of diseases is essential for appropriate treatment of patients, genetic counseling and prevention strategies. Characteristic features of patterns of inheritance are briefly reviewed and a short description of chromosomal disorders is also presented. In addition, applications of cytogenetic and molecular techniques and different types of mutations are discussed for genetic diagnosis of the pediatric genetic diseases.

use of reporter genes

Reporter genes, such as β-galactosidase and luciferase, have played vital roles in the understanding of the molecular mechanisms of gene expression.These reporter genes also have provided important information in the design of delivery systems in gene transfer into somatic tissue. However, monitoring these reporter systems in living animals requires either highly invasive tissue biopsy or post-mortem analysis of the animal under study. Optical imaging techniques, using GFP and luciferase, have been developed to provide non-invasive monitoring of gene expression in vivo, although these techniques are limited to either organisms which are transparent to light, or, in larger animals, to detection of gene expression near the surface of the skin.

Fluorescent proteins such as the “green fluorescent protein” (GFP) are popular tools in Caenorhabditis elegans, because as genetically encoded markers they are easy to introduce. Furthermore, they can be used in a living animal without the need for extensive sample preparation, because C. elegans is transparent and small enough so that entire animals can be imaged directly. Consequently, fluorescent proteins have emerged as the method of choice to study gene expression in C. elegans and reporter constructs for thousands of genes are currently available. When fused to a protein of interest, fluorescent proteins allow the imaging of its subcellular localization in vivo, offering a powerful alternative to antibody staining techniques. Fluorescent proteins can be employed to label cellular and subcellular structures and as indicators for cell physiological parameters like calcium concentration. Genetic screens relying on fluorescent proteins to visualize anatomical structures and recent progress in automation techniques have tremendously expanded their potential uses

Advantages of Green Fluorescent protein ( GFP)
There are three major contributions in biological science field by using GFP.
Protein tagging
GFP was first used to look into living cells by fluorescence microscopy to monitor protein localization and to visualize dynamic cellular events. A fusion between any cloned gene of interest and GFP can be produced by subcloning techinques and maybe introduced into the organism of interest by transient or stable expression.

Monitoring of gene expression
It allows the characterization of gene expression events at the single cell level. Robinett use the tight and specific binding properties between lac operator and lac repressor to detect the incorporated site in living cells by lac repressor-GFP fusion protein.

Biological screens
Because the GFP signal can be detected easily and rapidly without manipulation of the sample, perhaps the most promising emerging use of GFP is in selection procedures and in drug and genetic screens. Screening of living cells is especially important in the selection of embryonic stem cells and the production of transgenic animals.
Injection of GFP-labeled tumor-forming cells into nude mice not only labels the tumor, but also allows detection of micrometatastases in locations distant from the primary tumor. This mouse model can now be used both for the study of tumor progression and as an assay system in the search for compounds interfering with metastasis.
GFP will also be useful in drug screens. An impressive example is the establishment of cell lines carry GFP under the control of the HIV-1 long-terminal repeat. Infection of these normally non-fluorescent cells results in the appearance of the GFP signal in the cell due to the transcriptional activation of the HIV-GFP reporter.

Expressed fusion proteins are generally not toxic to cells.
Importantly, detection does not require fixation or permeabilization of cells; therefore, compareed with immnuocytochemistry techniques using fixed cells, the likelihood of artifacts is reduced.
Its tight, barrel-like structure protects the overall conformation of GFP protein and ensures that attachment of even large protein moieties does not affect its fluorescence capacity.

Limitations of GFP:
It is necessary that each fusion protein be tested for its functionality in vivo because GFP-tag is so relatively large that affect the function of fused protein of interest.
The GFP signal can not be amplified in a controlled manner, possibility preventing detection of low expression levels.
In addition, folding of GFP into its active, fluorescent form is fairly slow and occurs in the order of hours. This makes the study of the fast transcriptional activation processes difficult.

Advantages of Genetic mutant analysis

Genetic testing has potential benefits whether the results are positive or negative for a gene mutation. Test results can provide a sense of relief from uncertainty and help people make informed decisions about managing their health care. For example, a negative result can eliminate the need for unnecessary checkups and screening tests in some cases. A positive result can direct a person toward available prevention, monitoring, and treatment options. Some test results can also help people make decisions about having children. Newborn screening can identify genetic disorders early in life so treatment can be started as early as possible.

Limitations of Genetic mutant analysis. This test analyzes only certain important gene(s) associated with certain hereditary cancer risks. Genetic testing clarifies cancer risks for only those cancers related to the genes analyzed. If you are found to be a carrier of a gene that predisposes you to cancer, there may be differing opinions among physicians about the best steps to take. Y