GENE MANIPULATION

Gene Manipulation



Gene Manipulation

Answer of Question No. 1(a)

The goal of modern molecular cell biology is nothing short of understanding the biochemical, cellular, and organismal functions of all the proteins encoded in the genome. In the preceding sections, we have discussed the isolation and analysis of mutants, the genetic mapping of mutations, and finally the isolation and cloning of mutation-defined genes. This approach can provide valuable information about the molecular mechanisms underlying the cellular processes affected by the original mutations and the in vivo functions of the normal proteins encoded by the affected genes.

However, many genes have been identified based on the biochemical properties of their encoded protein, the sequence similarity of the encoded protein with proteins of known function, or their interesting patterns of expression in development. In the absence of mutant forms of such genes, their in vivo functions may be unclear. By mutating a specific gene in vitro and then replacing the normal copy in the genome with a mutant form, scientists can assess its in vivo function. The process of isolating normal genes to be mutated will be greatly simplified as sequencing of the genomes of several model organisms and of the human genome progresses. Whether starting from a normal protein or sequenced genome, this approach can be summarized as follows:

Other techniques permit the introduction of foreign genes or altered forms of an endogenous gene into an organism.

Answer of Question No. 1(b)

For the most part, these techniques do not result in replacement of the endogenous gene, but rather in the integration of additional copies of it. Such introduced genes are called transgenes; the organisms carrying them are referred to as transgenics. Transgenes can be used to study organismal function and development in a variety of different ways. For instance, genes that are normally expressed at specific times and places during development can be genetically engineered in vitro to be expressed in different tissues at different times and then reintroduced into the animal to assess the cellular and organismal consequences. For example, the Antennapedia (Antp) gene in Drosophila normally controls leg development, but misexpression of this gene in the developing antenna transforms it into a leg.

Answer of Question No. 1(c)

The production of both gene-targeted knockout and transgenic animals makes use of techniques for mutagenizing cloned genes in vitro and then transferring them into eukaryotic cells. We briefly describe these procedures first, and then discuss the production and uses of knockout and transgenic organisms. Specific sequences in cloned genes can be altered in vitro and then introduced into experimental organisms. This approach has been exploited primarily to study two questions. First, what is the relationship between the structure of a particular protein and its biological function? And second, what is the specific DNA sequences required to determine the expression pattern of a particular gene? A variety of enzymatic and chemical methods are available for producing site-specific mutations in vitro. In recent years, however, the most common methods use specific oligonucleotides as ...