遗传学实验技术

Random Chimeragenesis on Transient Templates (RACHITT) has been used to create libraries averaging 12 (1) or even 19 (L. Encell, unpublished) crossovers per gene in a single round of gene family shuffling. RACHITT creates chimeric genes by aligning parental gene “donor” fragments on a full-l ...

In vitro polymerase chain reaction (PCR)-based recombination methods are used to shuffle segments from various homologous DNA sequences to produce highly mosaic chimeric sequences. Genetic variations created in the laboratory or existing in nature can be recombined to generate l ...

The production of transgenic mice using small DNA constructs has been widely used for many years to investigate the regulation of gene activity. Small plasmid-based constructs (less than 20 kb) have been favored for a number of reasons, particularly the ease with which they can be manipulated and ...

Experimental studies that investigate the functional and mechanistic properties of an imprinted locus require material in which the two parental chromosome homologs can be easily distinguished. The use of animals with uniparental duplications and deficiencies of imprinted r ...

It is now generally believed that DNA methylation is responsible for genomic imprinting in mammals (1). Recent experimental evidence has provided an elegant mechanism for repression of gene expression by DNA methylation. This evidence suggests that proteins that recognize specif ...

DNA methylation has long been associated with stable transcriptional silencing and a repressive chromatin structure (reviewed in refs. 1,2). Differential methylation is associated with imprinting, carcinogenesis, silencing of repetitive DNA, and allows for differentiat ...

The acetylation of core histones can modulate the expression of numerous genes. In general, the deacetylation of histones results in transcriptional repression, whereas increases in histone acetylation lead to the enhancement of gene transcription. Since histone acetylation is ...

To further our understanding of genomic imprinting it will be essential to identify key control elements, and to investigate their regulation by both epigenetic modifications (such as DNA methylation) and trans-acting factors. So far, sequence elements that regulate parental alle ...

The first imprinted genes were identified in the early 1990s (e.g., refs. 1 1,2) and there are now over 40 mammalian genes known to be regulated by genomic imprinting (for an up-to-date list, see ref. 3). The details of the mechanism that discriminates between the active and silent alleles of these genes, bas ...

DNA methylation at position 5 in the cytosine ring in the sequence CpG can be detrimental to the transcription of a variety of genes in higher eukaryotes (1,2). Although the significance of this transcriptional repression is currently under debate (3,4), there is little disagreement that it pla ...

DNA methylation is a possible candidate for a genomic imprinting marker in mammals. This epigenetic modification of DNA satisfies several essential criteria for the identification of the parental origin of individual alleles and larger portions of the genome: DNA methylation is stab ...

Genomic imprinting is an epigenetically controlled form of gene regulation leading to the preferential expression of one parental gene copy. To date, approximately 40 imprinted genes have been described that are exclusively or predominantly expressed from either the paternal or the ...

Embryonic stem (ES) cells are a cell culture derivative of the blastocyst inner cell mass (ICM), the latter giving rise to the embryo, the amnion, the yolk sac, and the chrorioallantoic portion of the placenta. Blastocyst injection chimera experiments show that ES cells are similar to early-stage ...

DNA methylation is a mechanism for regulation of gene expression in animals (1-3). The addition of a methyl group at the 5-position of cytosine bases occurs exclusively at CpG dinucleotides. CpG dinucleotides in the vertebrate genome are underrepresented and amount to 1% of the genome (4). Howev ...

Methylation of the cytosine base in cytosine-guanine (CG) dinucleotides of genomic DNA is likely to be one of the primary epigenetic “imprints” that results in parental allele-specific expression of certain mammalian genes (1). Despite the increasing popularity of bisulphite seque ...

Chromosome replication banding studies show that homologous regions on a pair of autosomes generally replicate at the same time in S phase (1). Izumikawa et al. first observed that this was not the case for the imprinted chromosomal region 15q11-q13 (2). This observation has been confirmed in oth ...

One of the difficulties associated with the analysis of imprinted gene expression is the need to distinguish RNA synthesis occurring at the maternal vs the paternally inherited copy of the gene. Most of the techniques used to examine allele-specific expression exploit naturally occurr ...

An unexpected outcome of the diploid genome is that evolutionary strategies have evolved to express only one of the alleles (1). The rapidly expanding list of genes that are expressed monoallelically fall into three main categories: random inactivation, allelic exclusion, and genomic i ...

Imprinted genes show monoallelic expression from either the paternal or maternal genome (1,2), and their regulated expression is usually associated with the existence of parentally differentially methylated regions on genomic DNAs (3,4). Because of this, essentially two differ ...

The technique of representational difference analysis (RDA) was originally described by Lisitsyn et al. (1993) as a means of identifying differences between complex genomes by the use of subtractive hybridisation (1). This protocol for methylation-sensitive RDA (Me-RDA) describ ...