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Recombinase-mediated Cassette Exchange
RMCE (recombinase-mediated cassette exchange) is a procedure in reverse genetics allowing the systematic, repeated modification of higher eukaryotic genomes by targeted integration, based on the features of site-specific recombination processes (SSRs). For RMCE, this is achieved by the clean exchange of a preexisting gene cassette for an analogous cassette carrying the "gene of interest" (GOI). The genetic modification of mammalian cells is a standard procedure for the production of correctly modified proteins with pharmaceutical relevance. To be successful, the transfer and expression of the transgene has to be highly efficient and should have a largely predictable outcome. Current developments in the field of gene therapy are based on the same principles. Traditional procedures used for transfer of GOIs are not sufficiently reliable, mostly because the relevant epigenetic influences have not been sufficiently explored: transgenes integrate into chromosomes with low efficiency and ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Reverse Genetics
Reverse genetics is a method in molecular genetics that is used to help understand the function(s) of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proceeds in the opposite direction to forward genetic screens of classical genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes are controlled by particular genetic sequences. Automated DNA sequencing generates large volumes of genomic sequence data relatively rapidly. Many genetic sequences are discovered in advance of other, less easily obtained, biological information. Reverse genetics attempts to connect a given genetic sequence with specific effects on the organism. Reverse genetics systems can also allow the recovery and generation of infectious or defective viruses with desired mutations. This allows the ability to study the virus ''in vitro'' and ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Deletion (genetics)
In genetics, a deletion (also called gene deletion, deficiency, or deletion mutation) (sign: Δ) is a mutation (a genetic aberration) in which a part of a chromosome or a sequence of DNA is left out during DNA replication. Any number of nucleotides can be deleted, from a single base to an entire piece of chromosome. Some chromosomes have fragile spots where breaks occur which result in the deletion of a part of chromosome. The breaks can be induced by heat, viruses, radiations, chemicals. When a chromosome breaks, a part of it is deleted or lost, the missing piece of chromosome is referred to as deletion or a deficiency. For synapsis to occur between a chromosome with a large intercalary deficiency and a normal complete homolog, the unpaired region of the normal homolog must loop out of the linear structure into a deletion or compensation loop. The smallest single base deletion mutations occur by a single base flipping in the template DNA, followed by template DNA strand sli ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Applied Genetics
Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. New DNA is obtained by either isolating and copying the genetic material of interest using recombinant DNA methods or by artificially synthesising the DNA. A construct is usually created and used to insert this DNA into the host organism. The first recombinant DNA molecule was made by Paul Berg in 1972 by combining DNA from the monkey virus SV40 with the lambda virus. As well as inserting genes, the process can be used to remove, or "knock out", genes. The new DNA can be inserted randomly, or targeted to a specific part of the genome. An organism that is generated through genetic engineering is considered to be genetically modified (GM) ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Homologous Recombination
Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in cellular organisms but may be also RNA in viruses). Homologous recombination is widely used by cells to accurately DNA repair harmful breaks that occur on both strands of DNA, known as double-strand breaks (DSB), in a process called homologous recombinational repair (HRR). Homologous recombination also produces new combinations of DNA sequences during meiosis, the process by which eukaryotes make gamete cells, like sperm and egg cells in animals. These new combinations of DNA represent genetic variation in offspring, which in turn enables populations to adapt during the course of evolution. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses. Horizon ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Cre-Lox Recombination
Cre-Lox recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems. The Cre-lox recombination system has been particularly useful to help neuroscientists to study the brain in which complex cell types and neural circuits come together to generate cognition and behaviors. NIH Blueprint for Neuroscience Research has created several hundreds of Cre driver mouse lines which are currently used by the worldwide neuroscience community. The system consists of a single enzyme, Cre recombinase, that recombines a pair of short target sequences called the ''Lox'' sequences. This system can be implemented without inserting any extra supporting proteins or sequences. The Cre enzyme and the original ''Lox'' sit ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Cre Recombinase
Cre recombinase is a tyrosine recombinase enzyme derived from the P1 bacteriophage. The enzyme uses a topoisomerase I-like mechanism to carry out site specific recombination events. The enzyme (38kDa) is a member of the integrase family of site specific recombinase and it is known to catalyse the site specific recombination event between two DNA recognition sites ( LoxP sites). This 34 base pair (bp) loxP recognition site consists of two 13 bp palindromic sequences which flank an 8bp spacer region. The products of Cre-mediated recombination at loxP sites are dependent upon the location and relative orientation of the loxP sites. Two separate DNA species both containing loxP sites can undergo fusion as the result of Cre mediated recombination. DNA sequences found between two loxP sites are said to be " floxed". In this case the products of Cre mediated recombination depends upon the orientation of the loxP sites. DNA found between two loxP sites oriented in the same direction w ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Site-specific Recombination
Site-specific recombination, also known as conservative site-specific recombination, is a type of genetic recombination in which DNA strand exchange takes place between segments possessing at least a certain degree of sequence homology. Enzymes known as site-specific recombinases (SSRs) perform rearrangements of DNA segments by recognizing and binding to short, specific DNA sequences (sites), at which they cleave the DNA backbone, exchange the two DNA helices involved, and rejoin the DNA strands. In some cases the presence of a recombinase enzyme and the recombination sites is sufficient for the reaction to proceed; in other systems a number of accessory proteins and/or accessory sites are required. Many different genome modification strategies, among these recombinase-mediated cassette exchange (RMCE), an advanced approach for the targeted introduction of transcription units into predetermined genomic loci, rely on SSRs. Site-specific recombination systems are highly specific, fa ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Enhancer (genetics)
In genetics, an enhancer is a short (50–1500 bp) region of DNA that can be bound by proteins ( activators) to increase the likelihood that transcription of a particular gene will occur. These proteins are usually referred to as transcription factors. Enhancers are ''cis''-acting. They can be located up to 1 Mbp (1,000,000 bp) away from the gene, upstream or downstream from the start site. There are hundreds of thousands of enhancers in the human genome. They are found in both prokaryotes and eukaryotes. The first discovery of a eukaryotic enhancer was in the immunoglobulin heavy chain gene in 1983. This enhancer, located in the large intron, provided an explanation for the transcriptional activation of rearranged Vh gene promoters while unrearranged Vh promoters remained inactive. Locations In eukaryotic cells the structure of the chromatin complex of DNA is folded in a way that functionally mimics the supercoiled state characteristic of prokaryotic DNA, so although the e ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Insulator (genetics)
An insulator is a type of cis-regulatory element known as a long-range regulatory element. Found in multicellular eukaryotes and working over distances from the promoter element of the target gene, an insulator is typically 300 bp to 2000 bp in length. Insulators contain clustered binding sites for sequence specific DNA-binding proteins and mediate intra- and inter-chromosomal interactions. Insulators function either as an enhancer-blocker or a barrier, or both. The mechanisms by which an insulator performs these two functions include loop formation and nucleosome modifications. There are many examples of insulators, including the CTCF insulator, the ''gypsy'' insulator, and the β-globin locus. The CTCF insulator is especially important in vertebrates, while the ''gypsy'' insulator is implicated in ''Drosophila.'' The β-globin locus was first studied in chicken and then in humans for its insulator activity, both of which utilize CTCF. The genetic implications of insulat ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Multiplexing Recombinase-mediated Cassette Exchange
In telecommunications and computer networking, multiplexing (sometimes contracted to muxing) is a method by which multiple analog or digital signals are combined into one signal over a shared medium. The aim is to share a scarce resource - a physical transmission medium. For example, in telecommunications, several telephone calls may be carried using one wire. Multiplexing originated in telegraphy in the 1870s, and is now widely applied in communications. In telephony, George Owen Squier is credited with the development of telephone carrier multiplexing in 1910. The multiplexed signal is transmitted over a communication channel such as a cable. The multiplexing divides the capacity of the communication channel into several logical channels, one for each message signal or data stream to be transferred. A reverse process, known as demultiplexing, extracts the original channels on the receiver end. A device that performs the multiplexing is called a multiplexer (MUX), and a dev ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Stem Cells
In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type of cell in a cell lineage. They are found in both embryonic and adult organisms, but they have slightly different properties in each. They are usually distinguished from progenitor cells, which cannot divide indefinitely, and precursor or blast cells, which are usually committed to differentiating into one cell type. In mammals, roughly 50–150 cells make up the inner cell mass during the blastocyst stage of embryonic development, around days 5–14. These have stem-cell capability. ''In vivo'', they eventually differentiate into all of the body's cell types (making them pluripotent). This process starts with the differentiation into the three germ layers – the ectoderm, mesoderm and endoderm – at the gastrulation stage. However, whe ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Site-specific Recombinase Technology
Site-specific recombinase technologies are genome engineering tools that depend on recombinase enzymes to replace targeted sections of DNA. History In the late 1980s gene targeting in murine embryonic stem cells (ESCs) enabled the transmission of mutations into the mouse germ line, and emerged as a novel option to study the genetic basis of regulatory networks as they exist in the genome. Still, classical gene targeting proved to be limited in several ways as gene functions became irreversibly destroyed by the marker gene that had to be introduced for selecting recombinant ESCs. These early steps led to animals in which the mutation was present in all cells of the body from the beginning leading to complex phenotypes and/or early lethality. There was a clear need for methods to restrict these mutations to specific points in development and specific cell types. This dream became reality when groups in the USA were able to introduce bacteriophage and yeast-derived site-specific r ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
