Beyond seek and destroy: How to generate allelic series using genome editing tools

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECAS9 is a viable and efficient alternative, although it has a lower efficiency than SpCAS9 and LbCPF1 for creating KO mutations

Food distribution influences social organization and population growth in a small rodent

This is the postprint version of the article. The published article can be located at the publisher's websiteIn polygynous mammals, the spatial clumping and predictability of food should influence spacing behavior of females whose reproductive success depends to a great extent on food availability, which would in turn affect male spacing behavior. Changes in the social and mating systems can then influence individual fitness and population dynamics. To test these hypotheses, we manipulated food distribution and predictability in enclosed populations of bank voles (Myodes glareolus) and monitored spacing behavior, survival, and reproduction of adult females and males over 3 months. Food was either spread out (dispersed treatment), spatially clumped and highly predictable (clumped treatment) or spatially clumped but less predictable (variable treatment). We found that females in the clumped treatment were more aggregated and had more overlapping home ranges compared with females in the dispersed and variable treatments. Male spacing behavior followed the same patterns. Despite different social organizations between treatments, no differences in home range size and mating systems were found in females and males. In addition, we found that females in the clumped food treatment had a higher probability of successfully producing weaned offspring, likely due to lower infanticide rates. This led to higher population growth compared with the other 2 treatments. These results suggest a tight relationship between the spatiotemporal distribution of food, social organization, and population dynamics.2014-04-3

High pressure and high temperature in situ X-ray diffraction studies in the Paris-Edinburgh cell using a laboratory X-ray source

International audienceWe have developed a new laboratory experimental set-up to study in situ the pressure-temperature phase diagram of a given pure element or compound, its associated phase transitions, or the chemical reactions involved at high pressure and high temperature (HP-HT) between different solids and liquids. This new tool allows laboratory studies before conducting further detailed experiments using more brilliant synchrotron X-ray sources or before kinetic studies. This device uses the diffraction of X-rays produced by a quasi-monochromatic micro-beam source operating at the silver radiation (λ(Ag)Kα1,2 ≈ 0.56Å). The experimental set-up is based on a VX Paris-Edinburgh cell equipped with tungsten carbide or sintered diamond anvils and uses standard B-epoxy 5 or 7mm gaskets. The diffracted signal coming from the compressed (and heated) sample is collected on an image plate. The pressure and temperature calibrations were performed by diffraction, using conventional calibrants (BN, NaCl and MgO) for determination of the pressure, and by crossing isochores of BN, NaCl, Cu or Au for the determination of the temperature. The first examples of studies performed with this new laboratory set-up are presented in the article: determination of the melting point of germanium and magnesium under HP-HT, synthesis of MgB2 or C-diamond and partial study of the P, T phase diagram of MgH2

Food distribution influences social organization and population growth in a small rodent

This is the postprint version of the article. The published article can be located at the publisher's websiteIn polygynous mammals, the spatial clumping and predictability of food should influence spacing behavior of females whose reproductive success depends to a great extent on food availability, which would in turn affect male spacing behavior. Changes in the social and mating systems can then influence individual fitness and population dynamics. To test these hypotheses, we manipulated food distribution and predictability in enclosed populations of bank voles (Myodes glareolus) and monitored spacing behavior, survival, and reproduction of adult females and males over 3 months. Food was either spread out (dispersed treatment), spatially clumped and highly predictable (clumped treatment) or spatially clumped but less predictable (variable treatment). We found that females in the clumped treatment were more aggregated and had more overlapping home ranges compared with females in the dispersed and variable treatments. Male spacing behavior followed the same patterns. Despite different social organizations between treatments, no differences in home range size and mating systems were found in females and males. In addition, we found that females in the clumped food treatment had a higher probability of successfully producing weaned offspring, likely due to lower infanticide rates. This led to higher population growth compared with the other 2 treatments. These results suggest a tight relationship between the spatiotemporal distribution of food, social organization, and population dynamics.2014-04-3

Cells

Stem cells isolated from the apical papilla of wisdom teeth (SCAPs) are an attractive model for tissue repair due to their availability, high proliferation rate and potential to differentiate in vitro towards mesodermal and neurogenic lineages. Adult stem cells, such as SCAPs, develop in stem cell niches in which the oxygen concentration [O] is low (3-8% compared with 21% of ambient air). In this work, we evaluate the impact of low [O] on the physiology of SCAPs isolated and processed in parallel at 21% or 3% O without any hyperoxic shock in ambient air during the experiment performed at 3% O. We demonstrate that SCAPs display a higher proliferation capacity at 3% O than in ambient air with elevated expression levels of two cell surface antigens: the alpha-6 integrin subunit (CD49f) and the embryonic stem cell marker (SSEA4). We show that the mesodermal differentiation potential of SCAPs is conserved at early passage in both [O], but is partly lost at late passage and low [O], conditions in which SCAPs proliferate efficiently without any sign of apoptosis. Unexpectedly, we show that autophagic flux is active in SCAPs irrespective of [O] and that this process remains high in cells even after prolonged exposure to 3% O

Tissue engineering of the vascular system : from capillaries to larger blood vessels

Tissue engineering is a novel approach to the repair of wounded tissues. Application of this technology to the vascular system is important because of the fundamental nutritional role of the vasculature. This perspective is currently being applied to the first tissue-engineered organ: the skin. Knowledge of capillary constitution and factors inducing their formation has led to attempts to induce their formation in reconstructed skin. Such vascular conduits grown in vitro could also benefit the nutrition of tissues and organs in vivo. The paper reviews recent progress in thein-vitro development of vascularised skin and tissue-engineered blood vessels. It points out the necessity of obtaining pure and well-characterised cultures of the different cell populations that are the basic building blocks of the reconstructions. The importance of an adequate cell-culture environment (nutrients and bi-or tri-dimensional scaffolds for cells) for success in elaborating a reconstructed living tissue able to replace the original is emphasised. Engineered tissues can serve not only as tissue replacements but also asin-vitro models for research in organ physiology and physiopathology. These tissues are also attractive vehicles for gene therapy, one of the more promising new methods of disease treatment

A new differential thermal analysis setup for measuring high pressure phase transitions

International audienc

The self-assembly approach for organ reconstruction by tissue engineering

One must not forget that tissue engineering was first introduced as a life saving procedure for burn patients (1). The successful engraftment of autologous epidermal sheets was the first proof of concept of the powerful technology that we know today (2-4). However this very interesting initial approach fell into some disrepute because of perceived drawbacks and limitations (5, 6). The subsequent efforts in the field followed essentially three "schools" of thought. The first approach consists in the seeding of cells into various gels, which are then reorganized, by the incorporated cells (7-14). Alternatively, a second approach is to seed cells into a scaffold where they will thrive and secrete extracellular matrix (15-17). The scaffold materials are bioresorbable over a wide range of time periods depending on their chemical structures (18-25). A third approach is different since it uses the principle of a tissue template that allows, after implantation, the ingress of cells into the appropriately organized scaffold. Thus, these grafts are acellular and must stimulate the regenerative potential of the tissue wherever they are implanted (26-31). Our group has developed a different and original method for the reconstruction of soft tissues. It takes full advantage of the various intrinsic properties of cells when appropriately cultured. This entails particular media composition and appropriate mechanical straining of these threedimensional structures

A truly new approach for tissue engineering : the LOEX self-assembly technique

Tissue engineering has created several original and new avenues in the biomedical sciences. There is ongoing progress, but the tissue-engineering field is currently at a crossroads in its evolution; the validity of this technique is weIl established. Thus, new clinical applications must appear rapidly, within a few years, so that it will have a true impact on patient care. The self-assembly approach of the Laboratoire d'Organogénèse Expérimentale (LOEX) should be at the forefront