What is the ROSA26 gene?

ROSA26 is a locus used for constitutive, ubiquitous gene expression in mice. It was first isolated in 1991 in a gene-trap mutagenesis screen of embryonic stem cells (ESCs). Over 130 knock-in lines have been created based on the ROSA26 locus. The human homolog of the ROSA26 locus has been identified.

Why is ROSA26 used?

The Rosa26 locus is a useful place for inserting a gene, The location of the insertion is known — not random — and it allows scientists to study a gene without affecting the function of other genes. Knock-in models using this locus offer greater accuracy and reproducibility of results.

What is the ROSA26 promoter?

Rosa26 (Gt(ROSA)26Sor) was discovered in a gene-trap experiment by Philippe Soriano’s laboratory in 1991. The experimental strategy used a promoter-free retrovirus called “ROSABgal,” which encodes the beta galactosidase reporter. The retrovirus integrated randomly in the genome of mouse embryonic stem cells.

Where is ROSA26 located?

mouse chromosome 6
Known in scientific circles as Gt(ROSA)26Sor, the Rosa26 locus is a specific genetic site that is located on mouse chromosome 6, and is considered to be one of the ideal locations where knock-ins of interest can be targeted.

Do humans have a ROSA26 locus?

As in mice, they found that human ROSA26 was expressed in all adult tissues tested and in three different human ES cell lines2. The researchers then modified the widely used mouse Rosa26 vector and inserted a red fluorescent protein (RFP) gene into the locus in one of the human ES cell lines (HES 2).

What is the AAVS1 locus?

The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. The AAVS1 locus includes the coding region for myosin binding subunit 85 (MBS85).

What is loxP site?

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.

What is CreERT2?

CreERT2 encodes a Cre recombinase (Cre) fused to a mutant estrogen ligand-binding domain (ERT2) that requires the presence of tamoxifen for activity. We have previously shown that heterozygous Na(V)1.8-Cre mice will delete loxP flanked genes specifically in nociceptive sensory neurons from embryonic day 14.

What is knockin mouse model?

A Knockin mouse defines an animal model in which a gene sequence of interest is altered by one-for-one substitution with a transgene, or by adding gene sequences that are not found within the locus. The insertion of a transgene is typically done in specific loci.

What is the difference between sgRNA and crRNA?

sgRNA is an abbreviation for “single guide RNA.” As the name implies, sgRNA is a single RNA molecule that contains both the custom-designed short crRNA sequence fused to the scaffold tracrRNA sequence. sgRNA can be synthetically generated or made in vitro or in vivo from a DNA template.

Where is AAVS1?

chromosome 19
Adeno-associated virus integration site 1 is a viral integration site that in humans is encoded by the AAVS1 gene located on chromosome 19.

Is AAVS1 a gene?

AAVS1 is an exemplary locus within the PPP1R12C gene that permits robust expression of CAG promoter-driven transgenes.

What are floxed mice?

With the Cre-lox system, developed and perfected over the course of the 1990s and 2000s, floxed mice have become a major tool for genetic researchers. They use these mice to study, document and understand diseases and the possible cures and treatments they might develop in order to counter them.

Why is tamoxifen used in mice?

Tamoxifen is widely used for the induction of genomic recombination in mice (double-)transgenic for floxed genes and Tamoxifen specific estrogen receptors (ER) coupled to Cre-recombinase (supplemental Table 1) (Hayashi and McMahon 2002).

How are knockin mice created?

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.

What does floxed mouse mean?

The Cre-lox system allows for the deletion of a gene in mouse ES cells and its inversion or complete inactivation. This process is called “floxing” since it combines flanking the gene with the help of its adjacent two loxP sites and the Cre-lox recombination system.

What is cKO mice?

Conditional knockout (cKO) mice are genetically engineered animal models in which certain gene is inactivated in a specific tissue or on an inducible basis. Researchers can understand certain gene functions through time or tissue specific deletion to study the mechanisms of human disease.

Why target the ROSA26 locus for transgenic mice?

In transgenic mice, DNA is randomly integrated into the genome and the transgene copy number is variable. When targeting the Rosa26 locus, scientists achieve higher rates of success due to the known location and greater predictability of results compared to older transgene technology.

Why is ROSA26 used for Transgene expression?

In most cases, when Rosa26 is used, there’s a high rate of success when it comes to the correct insertion of the transgene and the facilitation of a stable, single-copy expression. Rosa26 also allows for a smaller number of mice because a targeted gene knock-in strategy is being used.

What is the ROSA26 mouse strain?

The ROSA26 mouse strain has one copy of the bacterial gene lacZ inserted into chromosome 6. The associated mouse promoter drives β ‐galactosidase expression in all adult tissues. Mice hemizygous for lacZ contain only one transgene allele.

Is ROSA26 a safe site for gene knockout in mouse models?

Given its wide expression across all cell types and developmental stages, the ROSA26 region is often used as a safe site for gene targeting in mouse models. Classifications of ROSA26 Gene Knockin Models