Simple route for nano-hydroxyapatite properties expansion.

Simple surface modification of nano-hydroxyapatite, through acid-basic reactions, allows expanding the properties of this material. Introduction of organic groups such as hydrophobic alkyl chains, carboxylic acid, and amide or amine basic groups on the hydroxyapatite surface systematically change the polarity, surface area, and reactivity of hydroxyapatite without modifying its phase. Physical and chemical properties of the new derivative particles were analyzed. The biocompatibility of modified Nano-Hap on Raw 264.7 cells was also assessed.

Targeted nucleotide exchange in bovine myostatin gene.

The myostatin gene, known as Growth Differentiation Factor 8 (GDF8), located at chromosome 2 (BTA2) in cattle, is specifically expressed during embryo development and in the adult skeletal muscle. Molecular analysis of this gene reveals the presence of three exons and two introns. Several cattle breeds, such as Piedmontese, Belgian Blue, Blond'Aquitaine, among others, show polymorphisms in this gene, which are directly related to double muscling phenotype. Piedmontese cattle shows a nucleotide transition G --> A (G938A) at exon 3, resulting in the substitution of cysteine to tyrosine, leading to a protein structure change, which determines myostatin inactivation and consequent muscular hypertrophy. The objective of this work was to implant the polymorphism G938A, naturally existent in Piedmontese breed, into in vitro propagated foetal myoblasts, from Nellore cattle. Single strand DNA (ssDNA) oligonucleotides were used to direct the same nucleotidic transition (G938A) to exon 3. Two transfection protocols (cationic lipid solution and electroporation) were tested and, 48 hours after transfection, RNA and DNA were extracted from myoblasts. Reverse transcription and polymerase chain reaction (PCR) were performed, using primers flanking the mutation region. The PCR products were cloned and analyzed by DNA sequencing, and it was possible to detect the nucleotidic CT transition at position 938, in the electroporated myoblasts. The existence of a positive signal in the transfection indicates that ssDNA oligonucleotides can be used to introduce this point mutation in specific functional gene sites.

Progress and prospects: targeted gene alteration (TGA).

Targeted gene repair or targeted gene alteration is a molecular strategy that aims to correct single base mutations responsible for genetic diseases. The concept involves using single-stranded DNA oligonucleotides to direct a nucleotide exchange reaction at the genomic site of the mutation. Investigators have made significant progress in elucidating the mechanism(s) by which the mutation is corrected and have begun to focus on several viable targets that show great potential for clinical application. During the past several years, the field has witnessed a phase transition as the focus has switched from purely basic science to a sustained translational mode. We highlight the important advances over the last two to three years, some of which have moved the technology closer to the clinic while some others have introduced new reasons for caution.

Gene therapy progress and prospects: targeted gene repair.

The capacity to correct a mutant gene within the context of the chromosome holds great promise as a therapy for inherited disorders but fulfilling this promise has proven to be challenging. However, steady progress is being made and the development of gene repair as a viable and robust approach is underway. Here, we present some of the recent advances that are helping to shape our thinking about the feasibility and the limitations of this technique. For the most part, these advances center on understanding the regulation of the reaction and validating its application in animal models.

Gene editing of a human gene in yeast artificial chromosomes using modified single-stranded DNA and dual targeting.

A single-nucleotide polymorphism (SNP) in a human gene can alter the behavior of the corresponding protein, and thereby affect an individual's response to drug therapy. Here, we describe a novel dual-targeting approach for introducing an SNP of choice into virtually any gene, through the use of modified single-stranded oligonucleotides (MSSOs). We use this strategy to create SNPs in a human gene contained in a yeast artificial chromosome (YAC). In the dual-targeting protocol, two different MSSOs are designed to edit two different bases in the same cell. A change in one of these genes is selective while the other is non-selective. We show that the population identified by selective pressure is enriched for cells that bear an edited base at the nonselective site. YACs with human genomic inserts containing particular SNPs or haplotypes can be used for pharmacogenomic applications, in cell lines and in transgenic animals.

Targeted gene repair in mammalian cells using chimeric RNA/DNA oligonucleotides and modified single-stranded vectors.

Determining the function of newly discovered genes is at the center of the evolving field of genomics. With the elucidation of the human DNA sequence, the importance of single base changes to gene function has become apparent. In some cases, nucleotide alteration accounts for inherited disorders, but in other cases, subtle, even conservative, base changes can influence the function of a gene and its product. To identify how critical genetic changes alter function, molecular tools such as synthetic vectors have been created to direct nucleotide exchange. Some of these vectors, including chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides, have shown promise in the specific alteration of a single base at an exact position within the gene. Here, we describe the activity of the synthetic vectors in a mammalian cell system. The episomal target contains a mutation in the neomycin resistance gene fused to a reporter ligand-binding domain. Correction of the mutated base enables translation of the normal fusion product. This protein can now bind a ligand, resulting in the expression of the fusion protein visualized by green fluorescence. Hence, the activity of any similar vector can be measured easily (and in real time) using confocal microscopy. The system provides the basis for examining the effectiveness of new targeting molecules for creating or repairing single base alterations. In addition, genes suspected of affecting the frequency of repair can be tested through their expression in cells harboring the mutated target plasmid. Once the frequency of exchange in cells is established, the use of these vectors will become commonplace in a process designed to generate specific single base changes in genes involved in signal transduction. Such changes should help define functional domains within these proteins.