Cre recombinase resources for conditional mouse mutagenesis

Large scale international activities for systematic conditional mouse mutagenesis, exploiting advances in the sophisticated manipulation of the mouse genome, has established the mouse as the premier organism for developing models of human disease and drug action. Conditional mutagenesis is critical for the elucidation of the gene functions that exert pleiotropic effects in a variety of cell types and tissues throughout the life of the animal. The majority of new mouse mutants are therefore designed as conditional, activated only in a specific tissue (spatial control) and/or life stage (temporal control) through biogenic Cre/loxP technologies. The full power of conditional mutant mice can therefore only be exploited with the availability of well characterized mouse lines expressing Cre-recombinase in tissue, organ and cell type-specific patterns, to allow the creation of somatic mutations in defined genes. This chapter provides an update on the current state of Cre driver mouse lines worldwide, and reviews the available public databases and portals that capture critical details of Cre driver lines such as the efficiency of recombination, cell tissue specificity, or genetic background effects. The continuously changing landscape of these mouse resources reflects the rapid progression of research and development in conditional and inducible mouse mutagenesis.     

Characterization of astrocyte-specific conditional knockouts

Conditional gene knockouts are a very powerful tool for elucidating gene function in animal physiology and behavior. To obtain cell-specific knockouts, a promoter is utilized that drives expression of Cre recombinase specifically to the cell population of interest. We describe several transgenic lines of mice that were created in an attempt to obtain astrocyte-specific gene recombination. A 2 kb fragment from the human glial fibrillary acidic protein promoter is utilized to drive expression of inducible Cre recombinase, with both the Tet-Off and tamoxifen responsive systems. We show data obtained from crosses with two Cre reporter lines, ROSA26R and an astrocyte Cre reporter created in our laboratory, to assess the cell specificity of gene recombination. Additionally, our system is shown to successfully recombine a floxed Connexin43 locus, although recombination is not as extensive as seen in crosses with reporter lines.     

Musashi1‐CreERT2: A new cre line for conditional mutagenesis in neural stem cells

The RNA‐binding protein Musashi1 (Msi1) is one of two mammalian homologues of DrosophilaMusashi, which is required for the asymmetric cell division of sensory organ precursor cells. In the mouse central nervous system (CNS), Msi1 is preferentially expressed in mitotically active progenitor cells in the ventricular zone (VZ) of the neural tube during embryonic development and in the subventricular zone (SVZ) of the postnatal brain. Previous studies showed that cells in the SVZ can contribute to long‐term neurogenesis in the olfactory bulb (OB), but it remains unclear whether Msi1‐expressing cells have self‐renewing potential and can contribute to neurogenesis in the adult. Here, we describe the generation of Msi1‐CreER^T2 knock‐in mice and show by cell lineage tracing that Msi1‐CreER^T2‐expressing cells mark neural stem cells (NSCs) in both the embryonic and adult brain. Msi1‐CreER^T2 mice thus represent a new tool in our arsenal for genetically manipulating NSCs, which will be essential for understanding the molecular mechanisms underlying neural development. genesis, 51:128–134, 2013. © 2012 Wiley Periodicals, Inc.     

EUCOMM--the European conditional mouse mutagenesis program.

Functional analysis of the mammalian genome is an enormous challenge for biomedical scientists. To facilitate this endeavour, the European Conditional Mouse Mutagenesis Program (EUCOMM) aims at generating up to 12 000 mutations by gene trapping and up to 8000 mutations by gene targeting in mouse embryonic stem (ES) cells. These mutations can be rendered into conditional alleles, allowing Cre recombinase-mediated disruption of gene function in a time- and tissue-specific manner. Furthermore, the EUCOMM program will generate up to 320 mouse lines from the EUCOMM resource and up to 20 new Cre driver mouse lines. The EUCOMM resource of vectors, mutant ES cell lines and mutant mice will be openly available to the scientific community. EUCOMM will be one of the cornerstones of an international effort to create a global mouse mutant resource.     

A mouse tool for conditional mutagenesis in ovarian granulosa cells

Here we describe the generation of an inducible Cre transgenic line allowing conditional mutagenesis in ovarian granulosa cells. We have expressed the tamoxifen inducible CreER^T2 fusion protein from a Bacterial Artificial Chromosome (BAC) containing the regulatory elements of the hydroxysteroid (17‐beta) dehydrogenase 1 (Hsd17b1) gene. Hsd17b1‐iCreER^T2 transgenic mice express the iCreER^T2 fusion protein exclusively in ovarian granulosa cells. Recombination analysis at the genomic DNA level using mice with “floxed” Stat3 alleles showed no Cre activity in absence of tamoxifen whereas tamoxifen treatment induced Cre activity solely in the ovaries. Further characterization of Hsd17b1‐iCreER^T2 mice using a Cre reporter line demonstrated that Cre‐mediated recombination was restricted to ovarian granulosa cells. Therefore, Hsd17b1‐iCreER^T2 mice should be a useful tool to analyze the gene functions in ovarian granulosa cells. genesis 48:612–617, 2010. © 2010 Wiley‐Liss, Inc.     

Male germline recombination of a conditional allele by the widely used Dermo1‐cre (Twist2‐cre) transgene

Conditional gene knockout using the Cre/loxP system is instrumental in advancing our understanding of the function of genes in a wide range of disciplines. It is becoming increasingly apparent in the literature that recombination mediated by some Cre transgenes can occur in unexpected tissues. Dermo1‐Cre (Twist2‐Cre) has been widely used to target skeletal lineage cells as well as other mesoderm‐derived cells. Here we report that Dermo1‐Cre exhibits spontaneous male germline recombination activity leading to a Cre‐mediated recombination of a floxed Ptk2 (Protein tyrosine kinase 2, also known as Fak [Focal adhesion kinase]) allele but not a floxed Rb1cc1 (RB1 inducible coiled‐coil 1, also known as Fip200 [FAK‐family Interacting Protein of 200 kDa]) allele at high frequency. This ectopic germline activity of Dermo1‐Cre occurred in all or none manner in a given litter. We demonstrated that the occurrence of germline recombination activity of Dermo1‐Cre transgene can be avoided by using female mice as parental Dermo1‐Cre carriers.     

Intersectional gene inactivation: there is more to conditional mutagenesis than Cre

正The efforts of many laboratories worldwide and large scale international mutagenesis consortia have resulted in mutagenesis of nearly all mouse genes, and present efforts are devoted to large scale phenotyping of germline null mutant mice (Ayadi et al., 2012). Although early lethality associated with loss of function of a given gene may prevent elucidating its function at a later stage or in a given tissue, conditional alleles in which a critical part of the gene is flanked by recombinase target sites provide an opportunity for addressing gene function at later stages (Branda and Dymecki,2004). Fortunately, many of the mutant alleles generated by the consortia allow for conditional mutagenesis using site directed recombinases.     

Adaptive mutagenesis accelerates microorganism evolution only while competing for resources: mathematic modeling

Mathematical simulation of adaptive mutagenesis was carried out. The results obtained indicate that adaptive mutagenesis can play an essential role in the survival of microbial populations. We show that the competition for natural resources is a necessary factor of adaptive mutagenesis.     

Beyond ecological traps: perceptual errors and undervalued resources

Ecological traps arise when animals choose to settle in poor-quality habitats, leading to a reduction in their survival or productivity. Although this phenomenon has received comprehensive theoretical treatment in the recent literature, the corollary of the ecological trap (when animals choose to avoid good-quality habitats) is rarely discussed. Failure to recognize high-quality sites could influence the ability of a population to reach its threshold size. An exploration of this phenomenon brings new insights into existing cases of ecological traps. By considering the full range of pitfalls faced during settlement, the relationship between habitat quality and population size might become clearer. We argue here that an improved understanding of factors determining the colonization of high-quality sites could aid conservationists in mitigating the damaging effects of maladaptive habitat selection.     

Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene

Transgenic tobacco plants were produced that contained single-copy pART54 T-DNA, with a 35S-uidA gene linked to loxP-flanked kanamycin resistance (nptII) and cytosine deaminase (codA) genes. Retransformation of these plants with pCre1 (containing 35S transcribed cre recombinase and hygromycin (hpt) resistance genes) resulted in excision of the loxP-flanked genes from the genome. Phenotypes of progeny from selfed-retransformed plants confirmed nptII and codA excision and integration of the cre-linked hpt gene. To avoid integration of the hpt gene, and thereby generate plants totally free of marker genes, we attempted to transiently express the cre recombinase. Agrobacterium tumefaciens (pCre1) was cocultivated with leaf discs of two pART54-transformed lines and shoots were regenerated in the absence of hygromycin selection. Nineteen of 773 (0.25%) shoots showed tolerance to 5-fluorocytosine (5-fc) which is converted to the toxic 5-fluorouracil by cytosine deaminase. 5-fc tolerance in six shoots was found to be due to excision of the loxP-flanked region of the pART54 T-DNA. In four of these shoots excision could be attributed to cre expression from integrated pCre1 T-DNA, whereas in two shoots excision appeared to be a consequence of transient cre expression from pCre1 T-DNA molecules which had been transferred to the plant cells but not integrated into the genome. The absence of selectable marker genes was confirmed by the phenotype of the T₁ progeny. Therefore, through transient cre expression, marker-free transgenic plants were produced without sexual crossing. This approach could be applicable to the elimination of marker genes from transgenic crops which must be vegetatively propagated to maintain their elite genotype.     

Development and use of a conditional antisense mutagenesis system in mycobacteria

Expression from a 2.3 kb region upstream of the inducible acetamidase gene from Mycobacterium smegmatis was shown to be upregulated by acetamide. A DNA fragment containing the start of the M. smegmatis hisD gene was cloned in front of the promoter, such that the antisense message was produced. When this construct was induced in vivo, the bacteria became phenotypically histidine auxotrophs; this auxotrophy was restored by histidine supplementation. Auxotrophy was not observed under non-induced conditions. Antisense mutagenesis may be useful for observing the phenotypic inactivation of specific mycobacterial genes, and an inducible system such as that described would allow the study of essential genes.     

FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei

We describe here a highly efficient procedure for conditional mutagenesis in Plasmodium. The procedure uses the site-specific recombination FLP-FRT system of yeast and targets the pre-erythrocytic stages of the rodent Plasmodium parasite P. berghei, including the sporozoite stage and the subsequent liver stage. The technique consists of replacing the gene under study by an FRTed copy (i.e., flanked by FRT sites) in the erythrocytic stages of a parasite clone that expresses the flip (FLP) recombinase stage-specifically--called the 'deleter' clone. We present the available deleter clones, which express FLP at different times of the parasite life cycle, as well as the schemes and tools for constructing new deleter parasites. We also outline and discuss the various strategies for exchanging a wild-type gene with an FRTed copy and for generating conditional gene knockout or knockdown parasite clones. Finally, we detail the protocol for obtaining sporozoites that lack a protein of interest and for monitoring sporozoite-specific DNA excision and depletion of the target protein. The protocol should allow the functional analysis of any essential protein in the sporozoite, liver stage or hepatic merozoite stages of rodent Plasmodium parasites.