遗传学实验技术

An end-trimming method was developed to amplify adjacent cDNA fragments by PCR (1,2). In an attempt to clone a novel cDNA, the 5′ and/or 3′ end are often missing. A well-known PCR technique for cloning the missing sequence is a method of rapid amplification of cDNA ends (RACE) (3). Although the missing 5′ and 3′ ends ...

The isolation of genomic 5′ regulating regions of genes starting from a suitable sequence (either a cDNA or an amino acid derived oligonucleotide) can be achieved in several ways. One of these, although long and labor-intensive, is the screening of a genomic library, followed by the isolation, sub ...

The SELEX (Systematic Evolution of Ligands by EXponential enrichments) combinatorial chemistry process is a procedure by which nucleic acid ligands of high affinity can be isolated against a molecular target (1). A wide variety of target molecules have been used successfully in the SELEX ...

Exon amplification is a technique designed to address a central problem in mammalian molecular genetics—how to extract coding sequences from large tracts of genomic DNA. As shown in Fig. 1, the technique (also known as exon trapping) exploits the ability of the eukaryotic splicing machinery ...

Isolation of a full-length gene on the basis of limited sequence information is often troublesome and challenging. Tremendous effort is needed to isolate a specific gene by screening cDNA or genomic libraries by oligonucleotide or nucleic acid probes. In those methods, basically, nucle ...

Since the first report on cDNA cloning in 1972 (1), this technology has developed into a powerful and universal tool in isolation, characterization, and analysis of both eukaryotic and prokaryotic genes. However, the conventional methods of cDNA cloning require much effort to generate a lib ...

We aimed to devise an appropriate method to directly link the fluorescence profile of chromosomal copy number alterations detected by chromosomal comparative genomic hybridization (cCGH) or any other hybridization or staining information with the genome sequence data. Our goal w ...

The ability to inhibit the activity of maternally stored gene products in Xenopus has led to numerous insights into early developmental mechanisms. Oocytes can be cultured and manipulated in vitro and then implanted into the body cavity of a host female to make them competent for fertilizati ...

The pipid frog Xenopus tropicalis has emerged as a powerful new model system for combining genetic and genomic analysis of tetrapod development with robust embryological, molecular, and biochemical assays. Its early development closely resembles that of its well-understood rela ...

The class II DNA “cut-and-paste” transposons have been used to efficiently modify the Xenopus genome for transgenesis applications. Once integrated, the transposon is an effective substrate for excision and re-integration (remobilization) elsewhere in the genome by simply supp ...

Here we present a protocol, which allows loss-of-function studies in Xenopus embryos using antisense morpholino oligonucleotides (MOs). Gene knockdown studies provide a critical method for assessing gene function in vitro and in vivo. Such studies are currently performed in Xenopus ...

Reverse genetics in Xenopus has been limited to knockdown strategies using antisense morpholino oligonucleotides (MOs). Recently, engineered zinc-finger nucleases have been used to induce targeted mutations resulting in null alleles. Zinc-finger nuclease (ZFN) technolo ...

Chemical genetics uses small molecules to modulate protein function and has the potential to perturb any biochemical event in a complex cellular context. The application of chemical genetics to dissect biological processes has become an attractive alternative to mutagenesis scr ...

Here we present a protocol for generating transgenic embryos in Xenopus laevis and Xenopus tropicalis. The method includes three steps: (1) The preparation of high-speed egg extracts, which facilitates the replacement of protamines in sperm nuclei with nucleosomes and decondenses t ...

Here we present a protocol for generating transgenic embryos in Xenopus using I-SceI meganuclease. This method relies on integration of DNA constructs, containing one or two I-SceI meganuclease sites. It is a simpler method than the REMI method of transgenesis, and it is ideally suited for gene ...

The two most common methods used to generate transgenic Xenopus embryos, restriction enzyme-mediated insertion, and I-SceI meganuclease take advantage of relatively common but spatially unpredictable double-stranded breaks in sperm, egg, or early embryo genomes. These metho ...

Identification of cis-regulatory elements, such as enhancers and promoters, is very important not only for analysis of gene regulatory networks but also as a tool for targeted gene expression experiments. In this chapter, we introduce an easy but reliable approach to predict enhancers of a g ...

The generation of transgenic animals is an essential tool for many genetic strategies. DNA “cut-and-paste” transposon systems can be used to efficiently modify the Xenopus genome. The DNA transposon substrate, harbored on a circularized plasmid, is co-injected into fertilized Xenop ...

Tissue-specific and inducible control of transgene expression is a cornerstone of modern studies in developmental biology. Even though such control of transgene expression has been accomplished in Xenopus, no general or widely available set of transgenic lines have been produced a ...

Chromatin immunoprecipitation (ChIP) is a powerful technique to study epigenetic regulation and transcription factor binding events in the nucleus. It is based on immune-affinity capture of epitopes that have been cross-linked to genomic DNA in vivo. A readout of the extent to which the ep ...