Academic literature on the topic 'Genetic recombination. Recombinant proteins. Recombinant DNA'

When a fragment of genomic DNA is introduced into a mammalian cell it can locate and recombine with the endogenous homologous sequences. This type of homologous recombination, known as gene targeting, is the subject of this chapter. Gene targeting has been widely used, particularly in mouse embryonic stem (ES) cells, to make a variety of mutations in many different loci so that the phenotypic consequences of specific genetic modifications can be assessed in the organism. The first experimental evidence for the occurrence of gene targeting in mammalian cells was made using a fibroblast cell line with a selectable artificial locus by Lin et al. (1), and was subsequently demonstrated to occur at the endogenous β-globin gene by Smithies et al. in erythroleukaemia cells (2). In general, the frequencies of gene targeting in mammalian cells are relatively low compared to yeast cells and this is probably related to, at least in part, a competing pathway: efficient integration of the transfected DNA into a random chromosomal site. The relative ratio of targeted to random integration events will determine the ease with which targeted clones are identified in a gene targeting experiment. This chapter details aspects of vector design which can determine the efficiency of recombination, the type of mutation that may be generated in the target locus, as well as the selection and screening strategies which can be used to identify clones of ES cells with the desired targeted modification. Since the most common experimental strategy is to ablate the function of a target gene (null allele) by introducing a selectable marker gene, we initially describe the vectors and the selection schemes which are helpful in the identification of recombinant clones (Sections 2-5). In Section 6, we describe the vectors and additional considerations for generating subtle mutations in a target locus devoid of any exogenous sequences. Finally, Section 7 is dedicated to the use of gene targeting as a method to express exogenous genes from specific endogenous regulatory elements in vivo, also known as ‘knock-in’ strategies. A targeting vector is designed to recombine with and mutate a specific chromosomal locus.

Over the past twenty-five years, genetic methods have generated a wealth of information on the regulation and the structure-function relationship of bacterial genes.These methods are based on the introduction of random mutations in a gene to alter its function. Subsequently, genetic techniques cure applied to localize the mutation, while the nature of the impairedfunction could be determined using biochemical methods. Classic examples of this approach is now considered to be the elucidation of the structure and function of genes, constituting the Escherichia coli lactose (lac) and tryptophan (

Human von Willebrand factor (vWF) is a plasma glycoprotein that is synthesized by endothelial cells and megakaryocytes, and perhaps by syncytiotrophoblast of placenta. The biosynthesis of vWF is very complex, involving proteolytic processing, glycosyla-tion, disulfide bond formation, and sulfation. Mature vWF consists of a single subunit of ∼ 250,000 daltons that is assembled into multimer ranging from dimers to species of over 10 million daltons. vWF performs its essential hemostatic function through several binding interactions, forming a bridge between specific receptors on the platelet sur

Haemophilia B, an X-linked recessive disease with an incidence of 1/30,000 newborn males, is due to defects in the gene for coagulation factor IX, which is on the long am of the X chromosome at band Xq27.1. This gene consists of approximately 34 Kb and contains 8 exons which specify a mRtfc of 2803 residues coding for a protein of 415 aa preceded by a prepro signal peptide of 46 aa. Coripanson of the functional domains of the factor IX protein with the exon structure of the gene supports the exon/protein domain hypothesis of gene evolution. The factor IX gene seems to be formed by a number of