GENERALIZED LACZ EXPRESSION WITH THE ROSA26 CRE REPORTER STRAIN PDF

Metrics details Abstract Cre-loxP based gene deletion approaches hold great promise to enhance our understanding of molecular pathways controlling mammary development and breast cancer. We reported earlier the generation of transgenic mice that express the Cre recombinase under the control of the mouse mammary tumor virus MMTV long terminal repeat LTR. These mice have become a valuable research tool to delete genes specifically in the mammary gland, other secretory organs, and the female germline. Our results show that MMTV-mediated Cre-activation is restricted to specific cell types of various secretory tissues and the hematopoietic system.

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ES cells harboring a single proviral copy were injected into blastocysts and mice containing the ROSA26 insertion were isolated. Hence, ES cells representing the original infection event were never produced and ROSA26 ES cells have had to be generated de novo from transgenic animals.

However ROSA26 mice appear to express lacZ in all hematopoietic cells as well as in all tissue of the embryos Zambrowicz et al,, It is thus very useful for a number of applications, including chimera analysis. Please note that for larger embryos E According to the tissue, penetration may be less than 1 mm.

Please consult the X-Gal staining protocol for how to proceed in such circumstances. The characterization of this locus has led to two methods for generating transgenic mice that express a transgene is a generalized fashion.

First, it is possible to use the genomic locus to target genes to the locus Soriano, Our ROSA26Cre reporter mice that express lacZ , available from the Jackson labs are readily recovered as fertile homozygotes, indicating that the difficulty in recovering GtROSA26 cannot be attributed to the mutation of the locus.

However in this case expression was broad but not uniform. Therefore in cases where uniform expression is desired, targeting to the locus might be the better way to go. Figure 1. The probe used for Southern blot analysis is shown as a shaded box. LoxP sites are indicated by black arrowheads. For more details, see Soriano, Targeting to the ROSA26 locus Figure 2 is conveniently achieved by introducing the desired gene into the first intron of the locus, at a unique XbaI site approximately bp upstream of the original gene trap line Figure 3.

A construct using the same splice acceptor as used in the original gene trap allele from adenovirus followed by the gene of interest and polyadenylation site is inserted at the unique XbaI site of pROSA A neomycin resistance cassette is included in the targeting vector.

Figure 2. Diagram of the ROSA26 locus. Sequence of the ROSA26 intron flanking the original provirus integration site. The XbaI site used for targeting genes to the locus is located bp upstream and is indicated in bold and underlined.

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The ubiquitous expression of ROSA26 in embryonic and adult tissues, together with the high frequency of gene-targeting events observed at this locus in murine ES cells has led to the establishment in the past 10 years of over knock-in lines expressing successfully from the ROSA26 locus a variety of transgenes including reporters, site-specific recombinases and, recently, noncoding RNAs. Different strategies can be employed to drive transgene expression from the ROSA26 locus. This chapter provides an overview of the current methodologies used to generate ROSA26 knock-in lines and describes different approaches that exploit the ROSA26 gene to control expression of transgenes, including miRNAs, in a temporal, cell-type, and stage-specific fashion. This process is experimental and the keywords may be updated as the learning algorithm improves. This is a preview of subscription content, log in to check access. Springer Nature is developing a new tool to find and evaluate Protocols.

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Generalized lacZ expression with the ROSA26 Cre reporter strain.

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