What is a neomycin cassette?
PGK–Neo (a hybrid gene consisting of the phosphoglycerate kinase I promoter driving the neomycin phosphotransferase gene) is a widely used cassette employed as a selectable marker for homologous recombination in embryonic stem cells.
What does Cre recombinase do?
Cre Recombinase is a Type I topoisomerase from bacteriophage P1 that catalyzes the site-specific recombination of DNA between loxP sites (1). The enzyme requires no energy cofactors and Cre-mediated recombination quickly reaches equilibrium between substrate and reaction products (2).
How do you make a KO mouse?
To produce knockout mice, researchers use one of two methods to insert artificial DNA into the chromosomes contained in the nuclei of ES cells. Both methods are carried out in vitro, that is in cultured cells grown in laboratory conditions.
Can Cre lox be used in vitro?
We have developed a novel way to use the Cre/loxP system for in vitro manipula- tion of DNA and a technique to clone DNA into circular episomes. The method is fast, reliable, and allows flexible cloning of DNA fragments into episomes containing a loxP site.
What are loxP sites?
LoxP sites are sites recognized by the bacteriophage cre recombinase, which excises sequences lying between two loxP sites leaving behind only one of the two sites.
How does CRE loxP work?
Concerning the mechanism of Cre-loxP system, a single Cre recombinase recognizes two directly repeated loxP site, then the Cre excises the loxP flanked (floxed) DNA, thus creating two types of DNA with circular, excised and inactivated gene Y (Figure 1A).
How do you make a LoxP mouse?
In theory, cKO mice can be generated by simultaneously inserting two loxP sites using two sgRNAs and two oligonucleotides as donors, but in practice the probability of obtaining cKO mice in one step is still very low, partly because the efficiency of oligo-mediated knockin is much lower than non-homologous end joining …
How do you make a knockout mutant?
Traditionally, homologous recombination was the main method for causing a gene knockout. 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.
What is double floxed?
The double-floxed inverse open reading frame (DIO) construct comprises inverted terminal repeats (ITR), the EF1α promoter, an eYFP-ChR2 fusion gene surrounded by a pair of LoxP sites and a pair of Lox2722 sites oriented inward, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a human …