Homologous Recombination Strategy Research Articles

Complete Deletion of All α-Dystrobrevin Isoforms Does Not Reveal New Neuromuscular Junction Phenotype

The dystrophin glycoprotein complex (DGC) is critical for muscle stability, and mutations in DGC proteins lead to muscular dystrophy. The DGC also contributes to the maturation and maintenance of the neuromuscular junction (NMJ). The gene encoding the DGC protein alpha-dystrobrevin undergoes alternative splicing to produce at least five known isoforms. Isoform-specific antibody staining and reverse transcription PCR in mutant mice with a deletion of exon 3 of the alpha-dystrobrevin gene suggested the existence of a remaining synaptic isoform, which might be compensating for alpha-dystrobrevin function. To test this possibility and to more completely understand the synaptic function of alpha-dystrobrevin, we used a two-step homologous recombination strategy combined with in vivo Cre-mediated excision to generate mice with a large deletion of the alpha-dystrobrevin gene to disrupt all isoforms. However, these mice did not exhibit a more severe NMJ phenotype than that observed in the exon 3-deleted mice. Nonetheless, these mice not only eliminate possible compensation by remaining isoforms of alpha-dystrobrevin, but also offer a conditional allele that could be used to identify tissue-specific and developmental functions of alpha-dystrobrevin. This work also demonstrates a successful strategy to achieve deletion of a large genomic sequence, which can be a valuable tool for functional studies of genes encoding multiple isoforms that span a large genomic region.

Multiple Strategies for Gene Transfer, Expression, Knockdown, and Chromatin Influence in Mammalian Cell Lines and Transgenic Animals

Manipulation of the eukaryotic genome has contributed to the progress in our knowledge of multicellular organisms but has also ameliorated our experimental strategies. Biological questions can now be addressed with more efficiency and reproducibility. There are new and varied strategies for gene transfer and sequence manipulation with improved methodologies that facilitate the acquisition of results. Cellular systems and transgenic animals have demonstrated their invaluable benefits. In this review, I present an overview of the methods of gene transfer with particular attention to cultured cell lines and large-scale sequence vectors, like artificial chromosomes, with the possibility of their manipulation based on homologous recombination strategies. Alternative strategies of gene transfer, including retroviral vectors, are also described and the applications of such methods are discussed. Finally, several comments are made about the influence of chromatin structure on gene expression. Recent experimental data have shown that for convenient stable transgene expression, the influence of chromatin structure should be seriously taken into account. Novel chromatin regulatory and structural elements are proposed as an alternative for proper and sustained gene expression. These chromatin elements are facing a new era in transgenesis and we are probably beginning a new generation of gene and cancer therapy vectors.

Histidine 129 in the 75-kDa Subunit of Mitochondrial Complex I from Yarrowia lipolytica Is Not a Ligand for [Fe4S4] Cluster N5 but Is Required for Catalytic Activity

Respiratory chain complex I contains 8-9 iron-sulfur clusters. In several cases, the assignment of these clusters to subunits and binding motifs is still ambiguous. To test the proposed ligation of the tetranuclear iron-sulfur cluster N5 of respiratory chain complex I, we replaced the conserved histidine 129 in the 75-kDa subunit from Yarrowia lipolytica with alanine. In the mutant strain, reduced amounts of fully assembled but destabilized complex I could be detected. Deamino-NADH: ubiquinone oxidoreductase activity was abolished completely by the mutation. However, EPR spectroscopic analysis of mutant complex I exhibited an unchanged cluster N5 signal, excluding histidine 129 as a cluster N5 ligand.

Rhesus monkey rhadinovirus (RRV): construction of a RRV-GFP recombinant virus and development of assays to assess viral replication

Rhesus monkey rhadinovirus (RRV) is a γ-2-herpesvirus that is closely related to Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8). Lack of an efficient culture system to grow high titers of virus, and the lack of an in vivo animal model system, has hampered the study of KSHV replication and pathogenesis. RRV is capable of replicating to high titers on fibroblasts, thus facilitating the construction of recombinant rhadinoviruses. In addition, the ability to experimentally infect naïve rhesus macaques with RRV makes it an excellent model system to study γ-herpesvirus replication. Our study describes, for the first time, the construction of a GFP-expressing RRV recombinant virus using a traditional homologous recombination strategy. We have also developed two new methods for determining viral titers of RRV including a traditional viral plaque assay and a quantitative real-time PCR assay. We have compared the replication of wild-type RRV with that of the RRV-GFP recombinant virus in one-step growth curves. We have also measured the sensitivity of RRV to a small panel of antiviral drugs. The development of both the recombination strategy and the viral quantitation assays for RRV will lay the foundation for future studies to evaluate the contribution of individual genes to viral replication both in vitro and in vivo.

Normal Neutrophil Function in Cathepsin G-Deficient Mice

Cathepsin G is a neutral serine protease that is highly expressed at the promyelocyte stage of myeloid development. We have developed a homologous recombination strategy to create a loss-of-function mutation for murine cathepsin G. Bone marrow derived from mice homozygous for this mutation had no detectable cathepsin G protein or activity, indicating that no other protease in bone marrow cells has the same specificity. Hematopoiesis in cathepsin G−/− mice is normal, and the mice have no overt abnormalities in blood clotting. Neutrophils derived from cathepsin G−/− mice have normal morphology and azurophil granule composition; these neutrophils also display normal phagocytosis and superoxide production and have normal chemotactic responses to C5a, fMLP, and interleukin-8. Although cathepsin G has previously shown to have broad spectrum antibiotic properties, challenges of mice with Staphylococcus aureus, Klebsiella pneumoniae, or Escherichia coli yielded survivals that were not different from those of wild-type animals. In sum, cathepsin G−/− neutrophils have no obvious defects in function; either cathepsin G is not required for any of these normal neutrophil functions or related azurophil granule proteases with different specificities (ie, neutrophil elastase, proteinase 3, azurocidin, and/or others) can substitute for it in vivo.

Normal Neutrophil Function in Cathepsin G-Deficient Mice

AbstractCathepsin G is a neutral serine protease that is highly expressed at the promyelocyte stage of myeloid development. We have developed a homologous recombination strategy to create a loss-of-function mutation for murine cathepsin G. Bone marrow derived from mice homozygous for this mutation had no detectable cathepsin G protein or activity, indicating that no other protease in bone marrow cells has the same specificity. Hematopoiesis in cathepsin G−/− mice is normal, and the mice have no overt abnormalities in blood clotting. Neutrophils derived from cathepsin G−/− mice have normal morphology and azurophil granule composition; these neutrophils also display normal phagocytosis and superoxide production and have normal chemotactic responses to C5a, fMLP, and interleukin-8. Although cathepsin G has previously shown to have broad spectrum antibiotic properties, challenges of mice with Staphylococcus aureus, Klebsiella pneumoniae, or Escherichia coli yielded survivals that were not different from those of wild-type animals. In sum, cathepsin G−/− neutrophils have no obvious defects in function; either cathepsin G is not required for any of these normal neutrophil functions or related azurophil granule proteases with different specificities (ie, neutrophil elastase, proteinase 3, azurocidin, and/or others) can substitute for it in vivo.

Transgenic mice in the analysis of reproductive development and function.

Transgenic mice have become important model systems for studying molecular, cellular, organ, and whole animal physiology. In particular, transgenic technology allows determination of cell lineage differentiation and the role(s) of specific proteins in mammalian development and oncogenesis. Transgenic mice can be created that express wild-type genes, mutant genes, marker genes or cell lethal genes in a tissue-specific manner. In addition, homologous recombination strategies in embryonic stem cells permit more sophisticated manipulation of the mammalian genome, including the functional deletion of specific genes in whole mice or in a specific tissue. Since all transgenic mice created must be bred to study the consequences of transgene or mutant allele expression, a number of effects of these genome manipulations on the reproductive development and function of these mice have been uncovered. In this review, we summarize the transgenic mouse models in which defects and abnormalities in the reproductive axis have been demonstrated.

A homologous recombination strategy to analyze the vinblastine resistance property of the V-circle in Leishmania

The generation of extrachromosomal DNA elements in Leishmania sp. can occur naturally or during in vitro selection with drugs. Previously, we had established a strong association between V-circle amplification and drug resistance in L. enriettii stepwise selected with increasing concentrations of vinblastine. Further, we demonstrated the presence of the lemdr1 gene in the amplified V-circle and subsequent functional analysis by transfection of this gene alone confirmed its role in conferring a drug-resistant phenotype, but the level of resistance was significantly lower than in cell lines obtained from stepwise drug selection. The aim of this work was to determine if other genes either on the V-circle or elsewhere in the genome were necessary for expression of vinblastine resistance. We report here the development of a homologous recombination method to convert the entire V-circle from the LeV160 cell line into a shuttle vector and further the targeted disruption of specific sites within the V-circle. Our results clearly demonstrate that the V-circle alone is sufficient to confer full vinblastine resistance and the disruption of the lemdr1 locus destroys the ability of the V-circle to confer vinblastine resistance.

Deletion analysis of the avermectin biosynthetic genes of Streptomyces avermitilis by gene cluster displacement.

Streptomyces avermitilis produces a group of glycosylated, methylated macrocyclic lactones, the avermectins, which have potent anthelmintic activity. A homologous recombination strategy termed gene cluster displacement was used to construct Neor deletion strains with defined endpoints and to clone the corresponding complementary DNA encoding functions for avermectin biosynthesis (avr). Thirty-five unique deletions of 0.5 to > 100 kb over a continuous 150-kb region were introduced into S. avermitilis. Analysis of the avermectin phenotypes of the deletion-containing strains defined the extent and ends of the 95-kb avr gene cluster, identified a regulatory region, and mapped several avr functions. A 60-kb region in the central portion determines the synthesis of the macrolide ring. A 13-kb region at one end of the cluster is responsible for synthesis and attachment of oleandrose disaccharide. A 10-kb region at the other end has functions for positive regulation and C-5 O methylation. Physical analysis of the deletions and of in vivo-cloned fragments refined a 130-kb physical map of the avr gene cluster region.