Understanding the Uses of the Rosa26 Knock-In Mouse

The Rosa26 locus in the mouse genome is a “safe harbor” which allows researchers to express genes of interest. Different gene targeting technologies (embryonic stem cells; CRISPR) are used to make specific DNA insertions at the Rosa26 locus. The rationale for scientists to use Rosa26 and other key benefits to using this type of mouse genetic model will be discussed.

What Is the Rosa26 Locus?
In the early 1990s, researchers isolated Rosa26, giving scientists a specific site for inserting genes to study. Prior to this, geneticists used transgenic mouse models to test hypotheses. Transgenic mice are generated by injecting plasmid DNA into a pronucleus. One limitation of transgenesis is that plasmid DNA randomly integrates into the mouse genome.

Scientifically referred to as GtROSA26, this locus is found on chromosome 6 of mice. It encodes a nonessential RNA that replicates throughout the body and every cell/tissue in the body that expresses it. Therefore, this locus provides a useful place for making gene insertions and studying how proteins impact the whole body.

Scientists initially created this line through embryonic stem cell retroviral gene trapping. Scientists identified embryonic stem cells that contained this as a proviral copy and injected them into blastocysts. Researchers separated the mice that had the Rosa26 insertion for future study. No cells had the vector Gen–ROSAβgeo natively, which required scientists to create the ones needed from scratch.

Prior to gene targeting, researchers created transgenic mice. This was accomplished by injecting plasmid DNA — transgene — into the mouse pronucleus. One disadvantage to this method is that the transgene randomly integrates into the mouse genome. In contrast, gene targeting allows scientists to either “knockout” a gene of interest or make an insertion — knock-in — at a specific site in the mouse genome. 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. Traditionally, these knock-in mice were generated using mouse embryonic stem cells, and this process has become more efficient with the advent of CRISPR technology. Biocytogen uses its proprietary CRISPR/Cas9-based Extreme Genome Editing (EGE™) system to obtain faster results by increasing the homologous recombination 10 to 20 fold.

Why Is Rosa26 Used?
Due to its ease of knocking in DNA, the Rosa26 locus on mouse chromosome 6 is very useful for scientists. Because this locus encodes a nonessential RNA and not a gene that serves a critical function, insertions lack adverse effects. The stable nature of the site and the ability for scientists to control global or conditional gene expression make the Rosa26 mouse model a versatile genetic tool.

1. Study Cell Lineages
   study cell lineages

By adding a reporter gene to this locus, researchers can trace cell lineages. The ability to follow genes throughout a line allows for a more in-depth study of how genetic code passes down and express themselves over generations.

Replacing the reporter with a toxin, scientists can ablate cell lineages. Knocking out genes is one method used to study how the absence of certain genes will affect an organism. Such a study becomes especially useful in identifying gene function through its absence.

1. Studying Gene Expression Throughout the Body
   The high level of expression of genes inserted into the Rosa26 site makes it desirable for researchers. A 1997 study of mice grown from embryonic stem cells infected with ROSAβgeo retrovirus showed expression throughout every tissue in the body.
2. Chimera Analysis
   Chimera analysis is another application of the Rosa26 mouse. Such studies examine mice from two zygotes that create animals with different genotypes in their cells. Some animals show expressions of those differing genotypes in their fur pattern or cells.

Rosa26 mice express β-galactosidase throughout their cells. As such, scientists can use Rosa26 cells as marked wild alleles. Crossing these cells with mutant cells marks those altered cells. These markers from the Rosa26 site can indicate different genotypes within a chimera.

1. Study Homozygotes Resulting From Knock-In
   While mice with combined genotypes prove useful for study, mice with singular genotypes from knocking in a gene at the Rosa26 site are also beneficial. In experiments using this locus, homozygotes produced remain alive, though few in number. The viability of these mice ensures the longer study of the results of the added gene at the Rosa26 location.
2. Examine Embryonic Cell Differentiation
   Using the Rosa26 site, scientists created stable cell lines that included protein kinase A, CA-PKA. When cells overexpressed PKA, they had greater differentiation and vascular formation. The researchers posited that inserting target genes at the Rosa26 site would allow them to better study how embryonic cells change into specific body cells.
3. Examine the Effects of Genes on Disease
   examine the effects of genes on disease

One reason scientists conduct Rosa26 knock-in studies is to see how a gene affects the development of a disease. Several diseases have suspected genetic links, and adding or subtracting these genes from the genome can determine if an individual gene or group of them play roles in the development of conditions such as diabetes or Alzheimer’s. For example, researchers used Rosa26 knock-in mice to examine how Met receptor tyrosine kinases (RTK) affected the onset of amyotrophic lateral sclerosis (ALS). That study found that increasing the Met RTK in mice did not have an effect on motor neuron development. However, it did slow the loss of motor neurons, delayed ALS onset and extended the lives of mice with ALS.

1. Studying the Location in Various Species
   Another useful aspect of this locus is its presence in different species. Although the Rosa26 locus was originally characterized in mice, it is also present in humans, pigs, rats, mice and rabbits. In 2018, researchers successfully identified the Rosa26 site in bovines. To prove the efficacy of Rosa26 as a safe harbor in bovines, researchers inserted genes into this locus and produced a cell line for use in future studies.
2. Study of Rosa26 Locus in Humans
   While studying gene insertions in mice could help future human genetics research, the use of Rosa26 in humans is not yet possible. Unlike the location in mice, Rosa26 in humans is close to critical genes. Gene editing the Rosa26 locus in humans could, therefore, disrupt the function of these other genes. Because the impact of adding genes at this site in humans could have unknown impacts, Rosa26 may not be a safe harbor insertion site in humans. However, AAVS1 is a safe harbor locus in the human genome.

Additional Benefits of Rosa26 Knock-In Mice
The Rosa26 locus offers several benefits over other locations on the genome, making it an ideal option for site-specific gene insertion.

1. Fewer Mice Needed
   Because scientists know the specific site for inserting genes, they require fewer mice for success. The need for fewer mice reduces the resources and time required, allowing further studies in other areas.
2. Higher Rates of Success
   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.
3. Stable Location
   Genetic insertions cannot be made at any position in the mouse genome, as some locations encode proteins with critical functions. In contrast, the Rosa26 locus is a safe harbor that will not disrupt gene function. Therefore, inserting genes with mutations or fluorescent reporters at the Rosa26 locus allows for the new gene to be expressed without interference.
4. Reproducibility of Results
   Numerous founder (F0 generation) mice are produced when a transgenic model is generated. These founders, though, had different genetic results, and they were almost impossible to reproduce. Part of this issue relates to the transgene copy number and loci differences in each model. For Rosa26, scientists have a distinct locus that allows them and other researchers to reproduce experimental results.
5. Express Target Gene After Cell Differentiation
   Express Target Gene After Cell Differentiation

Eventually, embryonic cells will differentiate into various body cells that will form the tissues, blood, organs, bones and other parts of the body. When using the Rosa26 site and inserting a target gene, the resulting expression of the gene appears in the cells after this differentiation event.

This widespread expression explains why even adult mice from this method of gene editing still show the traits of the mutation. The ability of the gene to track through various cell changes make it ideal for scientists looking to examine how cells alter through the life of the organism from fetus to adult.

How Do Scientists Create Rosa26 KI Mice?
Inserting genes into the Rosa26 locus can be accomplished either by using targeted embryonic stem cells or via the CRISPR/Cas9 system. A floxed sequence, which often contains neomycin, is positioned in front of a gene of interest to prevent it from transcribing and subsequently expressing.

For conditional expression, scientists employ the Cre-lox system. Crossing a conditional “floxed” mouse with a mouse expressing Cre recombinase deletes DNA sequences found within the floxed sequence. Without the LoxP-3XSTOP-LoxP upstream of the gene, the gene can now be transcribed. Until this Cre deleter removes the stop function, the cell will behave normally without expressing the gene. Using this conditional knock-in method, scientists can control when genes are expressed in different cells or tissues.

Typically, the inserted cassette also includes a reporter to track the gene’s expression. In many studies of this locus, scientists have used lacZ, a bacterial gene, as a reporter because unless integrated into exons or introns, it does not produce expression. When allowed to express itself, lacZ promotes β‐galactosidase expression in every adult tissue.

Improved use of CRISPR/Cas9 technology allows scientists to also use this method for knocking in genes at the Rosa26 site. Compared to older means of injecting zygotes, CRISPR created higher rates of success. Older methods only yielded 10% to 20% of live founder mutants, whereas CRISPR-produced mice had a 50% success rate with viability and mutation.

CRISPR RNA (crRNA) and TRACER RNA (tracrRNA) both bind together with each other and with the target gene sequence. The process requires crRNA to identify the DNA in the sequence while Cas9 proteins need tracrRNA for their activity.

To generate a knock-in mouse through CRISPR, crRNA, tracrRNA, Cas9, and a targeting vector are injected into the mouse zygote. The 2 RNAs guide the Cas9 nuclease to a specific site in the genome (e.g. Rosa26), and Cas9 makes the double-strand break. The cell will repair the broken DNA through a process called homology-directed repair (HDR). Genes of interest within a targeting vector become incorporated or inserted into the Rosa26 locus. Biocytogen’s EGE system speeds up HDR, therefore cutting down on the time to screen F0 mice.

To verify the accuracy of the results, Southern blot analysis provides the prime means of screening for random insertions. Because the targeting vector may produce random insertions in 32% of CRISPR projects, Southern blot analysis becomes a critical tool in testing the finished products. With this test, the position and copy number in the gene get verified for accuracy.

Why Use Rosa26 Mice in Your Research?
rosa26 mice for research

For research institutions that use mouse genetic models, the reliability of the results plays a crucial part in deciding where to source the animals. Verification of results, a long record of successes and numerous satisfied customers are signs of a quality source for Rosa26 mice and other genetic services.

Biocytogen can generate Rosa26 knock-in and conditional knock-in mice to help researchers address scientific questions. We are service providers and innovators. Our proprietary EGE method speeds HR for faster results without sacrificing accuracy. No core facility or vendor has this advantage.

Biocytogen offers a 100% satisfaction guarantee. For more information on our customized solutions or for inquiries into our Rosa26 mice creation process, contact us.