Non‐parallel recombination limits cre‐loxP‐based reporters as precise indicators of conditional genetic manipulation

Cre/LoxP‐mediated recombination allows for conditional gene activation or inactivation. When combined with an independent lineage‐tracing reporter allele, this technique traces the lineage of presumptive genetically modified Cre‐expressing cells. Several studies have suggested that floxed alleles have differential sensitivities to Cre‐mediated recombination, which raises concerns regarding utilization of Cre‐reporters to monitor recombination of other floxed loci of interest. Here, we directly investigate the recombination correlation, at cellular resolution, between several floxed alleles induced by Cre‐expressing mouse lines. The recombination correlation between different reporter alleles varied greatly in otherwise genetically identical cell types. The chromosomal location of floxed alleles, distance between LoxP sites, sequences flanking the LoxP sites, and the level of Cre activity per cell all likely contribute to observed variations in recombination correlation. These findings directly demonstrate that, due to non‐parallel recombination events, commonly available Cre reporter mice cannot be reliably utilized, in all cases, to trace cells that have DNA recombination in independent‐target floxed alleles, and that careful validation of recombination correlations are required for proper interpretation of studies designed to trace the lineage of genetically modified populations, especially in mosaic situations. genesis 51:436–442. © 2013 Wiley Periodicals, Inc.     

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel''s cartilage, nasal glands, tongue, and ear.     

C1188D mutation abolishes specific recognition between MLL1-CXXC domain and CpG site by inducing conformational switch of flexible N-terminal

Mixed lineage leukemia protein (MLL1 protein) recognizes the CpG site via its CXXC domain and is frequently associated with leukemia. The specific recognition is abolished by C1188D mutation, which also prevents MLL-related leukemia. In this paper, multiple molecular dynamic (MD) simulations were performed to investigate the mechanism of recognition and influences of C1188D mutation. Started from fully dissociated DNA and MLL1-CXXC domain, remarkably, the center of mass (COM) of MLL1-CXXC domain quickly concentrates on the vicinity of the CpG site in all 53 short MD simulations. Extended simulations of the wild type showed that the native complex formed in 500 ns among 4 of 53 simulations. In contrast, the C1188D mutant COM distributed broadly around the DNA and the native complex was not observed in any of the extended simulations. Simulations on the apo MLL1-CXXC domain further suggest that the wild type protein remained predominantly in an open form that closely resembles its structure in the native complex whereas C1188D mutant formed predominantly compact structures in which the N- terminal bends to D1188. This conformational switch hinders the formation of encounter complex, thus abolishes the recognition. Our study also provides clues to the study mechanism of recognition, by the CXXC domain from proteins like DNA methyltransferase and ten-eleven translocation enzymes.     

AN EVALUATION OF THE HYBRID SPECIATION HYPOTHESIS FOR XIPHOPHORUS CLEMENCIAE BASED ON WHOLE GENOME SEQUENCES

Once thought rare in animal taxa, hybridization has been increasingly recognized as an important and common force in animal evolution. In the past decade, a number of studies have suggested that hybridization has driven speciation in some animal groups. We investigate the signature of hybridization in the genome of a putative hybrid species, Xiphophorus clemenciae, through whole genome sequencing of this species and its hypothesized progenitors. Based on analysis of this data, we find that X. clemenciae is unlikely to have been derived from admixture between its proposed parental species. However, we find significant evidence for recent gene flow between Xiphophorus species. Although we detect genetic exchange in two pairs of species analyzed, the proportion of genomic regions that can be attributed to hybrid origin is small, suggesting that strong behavioral premating isolation prevents frequent hybridization in Xiphophorus. The direction of gene flow between species is potentially consistent with a role for sexual selection in mediating hybridization.     

Variations in nitrogen use efficiency reflect the biochemical subtype while variations in water use efficiency reflect the evolutionary lineage of C4 grasses at inter‐glacial CO2

C₄ photosynthesis evolved multiple times in diverse lineages. Most physiological studies comparing C₄ plants were not conducted at the low atmospheric CO₂ prevailing during their evolution. Here, 24 C₄ grasses belonging to three biochemical subtypes [nicotinamide adenine dinucleotide malic enzyme (NAD‐ME), phosphoenolpyruvate carboxykinase (PCK) and nicotinamide adenine dinucleotide phosphate malic enzyme (NADP‐ME)] and six major evolutionary lineages were grown under ambient (400 μL L^?1) and inter‐glacial (280 μL L^?1) CO₂. We hypothesized that nitrogen‐related and water‐related physiological traits are associated with subtypes and lineages, respectively. Photosynthetic rate and stomatal conductance were constrained by the shared lineage, while variation in leaf mass per area (LMA), leaf N per area, plant dry mass and plant water use efficiency were influenced by the subtype. Subtype and lineage were equally important for explaining variations in photosynthetic nitrogen use efficiency (PNUE) and photosynthetic water use efficiency (PWUE). CO₂ treatment impacted most parameters. Overall, higher LMA and leaf N distinguished the Chloridoideae/NAD‐ME group, while NADP‐ME and PCK grasses were distinguished by higher PNUE regardless of lineage. Plants were characterized by high photosynthesis and PWUE when grown at ambient CO₂ and by high conductance at inter‐glacial CO₂. In conclusion, the evolutionary and biochemical diversity among C₄ grasses was aligned with discernible leaf physiology, but it remains unknown whether these traits represent ecophysiological adaptation.