遗传学实验技术

DNA shuffling is a method for in vitro recombination of homologous genes invented by W.P.C Stemmer (1). The genes to be recombined are randomly fragmented by DNaseI, and fragments of the desired size are purified from an agarose gel. These fragments are then reassembled using cycles of denaturati ...

Directed protein evolution is usually accomplished by generating random or targeted mutations in the protein coding sequence and screening the mutant proteins (library) for functional improvements. By targeting mutations to certain amino acids, we can map the enzyme active site, in ...

Random oligonucleotide mutagenesis is a method to generate diversity that consists of incorporating random mutations, encoded on a synthetic oligonucleotide, into a specific region of a gene. The number of mutations in individual enzymes within the population can be controlled by va ...

Random mutagenesis combined with high-throughput screening is a versatile strategy for improving protein functions or creating artificial enzymes (1,2). Several methods for introducing random mutations in vitro have been reported (3). Among these, error-prone PCR mutagenesi ...

The directed evolution of microorganisms requires the random introduction of mutations and subsequent selection of improved variants. Traditionally, mutagenesis has been performed using DNA-modifying chemicals or UV radiation. An alternative is the use of so-called mutator ...

A variety of methods have been developed for random mutagenesis of genes and whole plasmids to generate genetic diversity for directed evolution experiments (1). In particular, bacterial strains exhibiting unusually high rates of spontaneous DNA mutagenesis, or mutator strains, c ...

Directed evolution has become a powerful tool not only for improving the utility of enzymes in industrial processes, but also to generate variants that illuminate the relationship between enzyme sequence, structure, and function. The method most often used to generate variants with ran ...

Whole genes, plasmids, and even viral genomes can be assembled from relatively short, synthetic, overlapping oligodeoxyribonucleotides (oligos) by DNA polymerase extension (1,2). Once the initial investment in a set of oligos spanning the length of a gene has been made, a mutation can be gen ...

Genomic recombination is widely recognized as the principal mechanism by which proteins evolve new functions (1). Having realized the importance of recombination in evolution, scientists have developed a variety of methods to mimic this phenomenon in the lab to create libraries of gene ...

The process of preparing libraries of mutagenized or recombined gene sequences for screening or selection in Escherichia coli is a special application of cohesive-end subcloning (1). PCR products are digested with restriction endonucleases, ligated into an expression vector dig ...

Vector transport across the endothelium has long been regarded as one of the central “bottlenecks” in gene therapy research, especially as it pertains to the muscular dystrophies where the target tissue approaches half of the total body mass. Clinical studies of gene therapy for hemophilia B ...

Adeno-associated viral vector (AAV)-mediated gene transfer represents a promising gene replacement strategy for treating Duchenne muscular dystrophy (DMD). However, recent studies demonstrated cellular immunity specific to AAV capsid proteins in animal models, which re ...

The development of a nonhuman primate (NHP) model for vascular delivery of therapeutic transgenes with adeno-associated viral (AAV) vectors is crucial for successfully treating muscular dystrophies. Current animal models for Duchenne muscular dystrophy (DMD) gene therapy ha ...

Exon skipping is currently one of the most promising molecular therapies for Duchenne muscular �dystrophy (DMD). We have recently developed multiple exon skipping targeting exons 6 and 8 in �dystrophin mRNA of canine X-linked muscular dystrophy (CXMD), an animal model of DMD, which exhibi ...

Current strategies for managing congestive heart failure are limited, validating the search for an alternative treatment modality. Gene therapy holds tremendous promise as both a practical and translatable technology platform. Its effectiveness is evidenced by the improvem ...

Gene therapy of muscular dystrophy requires systemic gene delivery to all muscles in the body. Adeno-associated viral (AAV) vectors have been shown to lead to body-wide muscle transduction after a single intravascular injection. Proof-of-principle has been demonstrated in mouse mo ...

Cardiovascular disease is a major cause of morbidity and mortality in contemporary societies. While progress in conventional treatment modalities is making steady and incremental gains to reduce this disease burden, there remains a need to explore new and potentially therapeutic a ...

Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Prior strategies have employed cardio-pulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated vir ...

Female meiosis in Drosophila is interesting and exceptional in many respects. First, like all dipterans, Drosophila has a polytrophic type of ovary characterized by the presence of nurse cells, which provide ooplasma and yolk to developing oocytes, whereas meiotic chromosomes are met ...

Drosophila oogenesis is a fascinating phenomenon. The coordinated action of many cellular processes produces a fully mature egg containing a maternal dowry that both directs and supports development of the embryo. The ovary is one of the best studied organs of Drosophila, and much of our know ...