Sequence-specific modification of mouse genomic DNA mediated by gene targeting techniques

Abstract

The major impact of the human genome sequence is the understanding of disease etiology with deduced therapy. The completion of this project has shifted the interest from the sequencing and identification of genes to the exploration of gene function, signalling the beginning of the post-genomic era. Contrasting with the spectacular progress in the identification of many morbid genes, today therapeutic progress is still lagging behind. The goal of all gene therapy protocols is to repair the precise genetic defect without additional modification of the genome. The main strategy has traditionally been focused on the introduction of an expression system designed to express a specific protein, defective in the transfected cell. But the numerous deficiencies associated with gene augmentation have resulted in the development of alternative approaches to treat inherited and acquired genetic disorders. Among these one is represented by gene repair based on homologous recombination (HR). Simply stated, the process involves targeting the mutation in situ for gene correction and for restoration of a normal gene function. Homologous recombination is an efficient means for genomic manipulation of prokaryotes, yeast and some lower eukaryotes. By contrast, in higher eukaryotes it is less efficient than in the prokaryotic system, with non-homologous recombination being 10–50 fold higher. However, recent advances in gene targeting and novel strategies have led to the suggestion that gene correction based on HR might be used as clinical therapy for genetic disease. This site-specific gene repair approach could represent an alternative gene therapy strategy in respect to those involving the use of retroviral or lentiviral vectors to introduce therapeutic genes and linked regulatory sequences into random sites within the target cell genome. In fact, gene therapy approaches involving addition of a gene by viral or nonviral vectors often give a short duration of gene expression and are difficult to target to specific populations of cells. The purpose of this paper is to review oligonucleotide-based gene targeting technologies and their applications on modifying the mouse genome.

Delivery of Nucleic Acids and Gene Delivery

“…[7][8][9][10][11][12][13][14][15] The concept underlying antisense technology involves the use of a sequence that is complementary to a specific host mRNA, which inhibits this mRNA expression thereby inducing a blockade in the transfer of genetic information from DNA to protein. [39][40][41][42][43][44][45][46][47] Conversion of the mutations to the normal sequence (gene correction) and disruption (knockout) of disease-related genes would be achieved when the therapeutic ODN is delivered to the nucleus. 31,36 Many antisense sequences showed beneficial therapeutic effects in reducing tumor growth.…”

Comparative genetic analysis: the utility of mouse genetic systems for studying human monogenic disease

“…The exploitation of inducible promoters and tissue-specific recombinase enzymes also allows the deletion of a gene of interest in a particular organ, cell type, and/or stage of development; such conditional and tissue-specific knockouts provide more accurate and finely tuned systems to study gene function than those generated by conventional constitutive technology (Porret et al 2006 ). A combination of these approaches, in addition to the generation of multiple gene knockouts, has revolutionized the study of many fields of fundamental research, most significantly impacting developmental biology with major insights into the physiology of the hematopoietic, immune, skeletal, cardiovascular, and nervous systems (Shastry 1998 ; Sheahan et al 2004 ; Ning et al 2006 ).…”

“…This particularly applies to knockout mice of tumor suppressors which, with few exceptions, all show embryonic lethality with a distinctive pattern of organ malformations (Ghebranious and Donehower 1998 ). Although these mice have provided good models for the study of individual genes in embryonic development and the regulation of differentiation, apoptosis, and cell cycle control during organogenesis (Shastry, 1998 ), they have not necessarily been useful for the characterization of gene function in tumorigenesis. The development of conditional and tissue-specific gene-deficiency technologies mentioned above, however, has now overcome this restriction.…”

The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes

“…The former approach is aimed at the direct correction of genetic mutations, stably restoring the wild-type gene function in situ and maintaining gene regulation under the native sequence context. 1,2,3 Consequently, the targeted gene continues to be regulated by its endogenous machinery, maintaining its physiological expression pattern. 3 By contrast, the latter approach is based on the delivery of additional copies of the wild-type therapeutic gene, which is expressed under the control of strong exogenous promoters.…”