How does the CRE LOX system work?

How does the CRE LOX system work?

Cre recombinase proteins bind to the first and last 13 bp regions of a lox site forming a dimer. This dimer then binds to a dimer on another lox site to form a tetramer. The double stranded DNA is cut at both loxP sites by the Cre protein. The strands are then rejoined with DNA ligase in a quick and efficient process.

What is a conditional allele?

Conditional alleles in genetically modified mice allow for the deletion of a gene of interest in a target tissue when combined with a tissue-specific Cre recombinase. A conditional allele is achieved by introducing LoxP sites around a critical exon, a gene, or a cluster of genes.

How does gene knockout work?

This method involves creating a DNA construct containing the desired mutation. For knockout purposes, this typically involves a drug resistance marker in place of the desired knockout gene. This method then relies on the cell’s own repair mechanisms to recombine the DNA construct into the existing DNA.

What are conditional alleles?

Why is the CRE loxP system used in conditional knockout mice?

In particular, Cre-loxP system is widely used as an integral experimental tool for generating the conditional. This system has enabled researchers to investigate genes of interest in a tissue/cell (spatial control) and/or time (temporal control) specific manner.

What is CRE LOX used for?

The Cre/lox system is one of the most powerful and versatile tools developed for mouse genetics. It gives mouse researchers sophisticated control over the location and timing of gene expression. Cre/lox is usually used to make knockout alleles, but it can also be used to activate gene expression.

How do you make a CRE LOX mouse?

In this system, a region of interest flanked by two lox sites (floxed) is deleted or inverted by Cre-mediated recombination, leading to gene knockout only in a Cre-expressing cell. In general, Cre/lox mice are generated by mating a Cre-driver mouse with a flox mouse.

What is a stop cassette?

The STOP cassette consists of the 3′ portion of the yeast His3 gene, an SV40 polyadenylation sequence and a false translation initiation codon followed by a 5′ splice donor site.

What is a Floxed gene?

In genetics, floxing refers to the sandwiching of a DNA sequence (which is then said to be floxed) between two lox P sites. Floxing a gene allows it to be deleted (knocked out), translocated or inverted in a process called Cre-Lox recombination.

What is CRE dependent?

Cre-dependent gene expression – placing a stop codon with loxP sites on either side (often called a “lox-stop-lox” or “LSL” cassette) upstream of a gene of interest will prevent gene expression in the absence of Cre. In the presence of Cre, the stop codon is excised, and gene expression proceeds.

What is a Cre transgenic mouse?

A Cre mouse contains a Cre recombinase transgene under the direction of a tissue-specific promoter; a loxP mouse contains two loxP sites that flank a genomic segment of interest, the “floxed” locus. Typically, Cre and loxP mice are produced by using transgenic technology (Nagy 2000).

What does CRE stand for?

CRE, which stands for carbapenem-resistant enterobacteriaceae, are strains of bacteria that are resistant to carbapenem, a class of antibiotics typically used as a last resort for treating severe infections when other antibiotics have failed.

What are FRT sites?

FRT sites are used often (at least in Drosophila) for inducing deletions or “flipping out” of markers in transgenic constructs. When there are two FRTs sequences in tandem, after inducing flippase the DNA sequence that is between these two sites will be deleted.

How does FLP FRT work?

The Flp protein, much like Cre, is a tyrosine family site-specific recombinase. This family of recombinases performs its function via a type IB topoisomerase mechanism causing the recombination of two separate strands of DNA. Recombination is carried out by a repeated two-step process.

What is the full form of FLP?

Family Limited Partnership (FLP)

What is conservative site specific recombination?

Conservative site-specific recombination is a process that enables genetic recombination between DNA molecules that contain short DNA sequences, which are bound by specific recombinase proteins.

What are the three types of recombination?

At least four types of naturally occurring recombination have been identified in living organisms: (1) General or homologous recombination, (2) Illegitimate or nonhomologous recombination, (3) Site-specific recombination, and (4) replicative recombination.

How could transposons be used in the study of bacterial genetics?

Transposons can transfer from a plasmid to other plasmids or from a DNA chromosome to plasmid and vice versa that cause the transmission of antibiotic resistance genes in bacteria.

What are the two families of conservative site specific Recombinases?

Only two classes of conservative site-specific recombinases have been discovered so far: the invertase/resolvase family (also called the serine family) and the lambda integrase family (also called the tyrosine family).

What do you mean by recombination?

Recombination is a process by which pieces of DNA are broken and recombined to produce new combinations of alleles. This recombination process creates genetic diversity at the level of genes that reflects differences in the DNA sequences of different organisms.

What is replicative recombination?

General or homologous recombination occurs between DNA molecules of very similar sequence, such as homologous chromosomes in diploid organisms. The third type is replicative recombination, which generates a new copy of a segment of DNA.

Which of the following is not a recombination system?

Which of the following is NOT a recombination system? Explanation: Homologous recombination was first described in bacterial systems.

What are the characteristics of rough pneumococci strain?

1. What are the characteristics of rough pneumococci strain? Explanation: Griffith injected mice with a few rough (noncapsulated and nonpathogenic) pneumococci and a large number of heat-killed smooth cells.

What does a frequency of recombination of 50% indicate?

What does a frequency of recombination of 50% indicate? A) The genes are located on sex chromosomes. The two genes are likely to be located on different chromosomes.

What will happen if there were no genetic recombination during reproduction?

If genetic recombination did not occur during meiosis, breeding studies would show that the genes controlling certain traits are always inherited together, whereas others always are inherited independently; however, genetic recombination, or crossing over, results in the exchange of portions of homologous chromosomes …