Month: July 2020

Delivering nucleases in the form of mRNA is particularly attractive because it ensures that the potentially toxic nucleases are present only transiently. Moving beyond the issue of gene delivery, our study demonstrated proof of concept for the process of ex vivo gene therapy, wherein a patient’s cells are isolated, genetically modified in vitro and then reintroduced into the patient. Admittedly, challenges must be overcome before translation to the clinic. A feature critical to the success of ex vivo gene therapy is availability of a culturing method, something that had been established for rodent SSCs, but that is an area of fervent yet controversial investigation for human SSCs. Regarding correction of genes other than GFP, other examples of genome engineering at a model target locus have generally proven to be highly relevant in predicting applicability to new loci. In the case of genetic defects causing spermatogenic failure, corrected SSCs would have a selective advantage and may not need to be enriched before transplantation as only corrected SSCs would produce sperm. For diseases in which corrected cells do not have a selective advantage, strategies described above could facilitate enrichment of rare cells with the corrected loci. Many genetic diseases affect cells or tissues for which a cognate stem cell type is unknown and or impossible to culture. Many diseases also are systemic in nature, affecting numerous cell types that cannot be treated with a single cell type. Furthermore, even for certain diseases of the blood that have been successfully treated by genetic modification and transplantation of autologous hematopoietic stem cells, patients then live with the burden of potentially passing the heritable trait on to their children. Ultimately the most permanent and all-encompassing cure of a genetic disease would be to genetically modify the germ cells. The use of SSCs in our study is especially unique because unlike all other adult stem cell types, SSCs are capable of generating sperm, which carry genetic information to the next generation. SSCs are also unique in their developmental plasticity, capable of transdifferentiation to multiple lineages or even to pluripotency without the use of exogenous genetic factors. An obvious application one could imagine is treatment of infertility caused by mutations affecting germ cell development. However, several substantial issues would need to be addressed before applying germline gene therapy in humans. First, our transplantation study revealed that the gene-corrected cells appeared to R428 1037624-75-1 exhibit attenuated differentiation capability. The cause of this shortcoming is unclear. Previous studies showed that GS cells can be transfected and passaged extensively in vitro while still retaining the ability to produce spermatozoa following transplantation. Our data did not distinguish among potential effects of technical issues with the transplant recipients, genetic or epigenetic effects on the cells unrelated to genome editing, or unintended genomic changes related to off-target cutting by the ZFNs. In other experiments we have found that delivering ZFNs via DNA expression vectors causes gross chromosomal rearrangements.

For instance, one study showed that following transfection of 2.46108 cells with a targeting vector containing large homology arms, antibiotic selection and PCR screening led to the isolation of two targeted cell clones. We obtained a,0.1% gene correction rate using engineered nucleases. Thus, the induction of a site specific double-strand break increased the targeting frequency by.100,000 fold. The low rate of gene correction in SSCs may reflect inherent mechanisms of genome protection unique to germ cells; intrinsic differences in efficiency between cell types are not unexpected. Fortunately, one could invoke a strategy to enrich for corrected cells using multiple published methods. Enrichment strategies include FACS purification of transfected cells, the purification of cells that have undergone correction based on the modification of a surrogate reporter which dramatically enriches for modified cells, and the use of donor constructs and designs containing selectable markers that allow one to select for modified cells using the selectable marker and then subsequent “scarless” elimination of the selectable marker after identification. Thus, the low frequency of gene correction in SSCs does not preclude genome editing from being accomplished in this important stem cell type. The implications of our study are multi-faceted with applications in research and potentially ICG-001 therapeutics. Much progress remains to be made in understanding mechanisms controlling SSC fate, particularly in humans. The ability to make precise modifications to the genome could facilitate analysis of gene function, thereby advancing our understanding of SSCs and spermatogenesis. For instance, a point mutation identified in a genome-wide association study to be potentially associated with spermatogenic failure could be directly tested for functional importance using the technology demonstrated here. The ability to generate fluorescent reporters of gene expression by targeted addition is another potential research application. The relevance of these applications extends even beyond the study of SSCs, given that upon testicular transplantation of genetically engineered GS cells new transgenic mouse models can be generated. The implications of our study for medicine are two-fold. First, we addressed a pervasive challenge in gene therapy, namely gene delivery in a “hard to transfect” primary-like stem cell. Gene delivery is a particularly significant issue for nuclease-mediated gene correction because it is necessary to introduce three components into cells. In this study we demonstrated a gene delivery approach that may be widely applicable to other stem cells. In our preliminary experiments adeno-associated virus, and integration deficient lentivirus both were inadequate for accomplishing genome editing. Identification of a virus with the appropriate tropism for a cell type of interest and production of sufficient titers of infectious virus are among the complications of viral delivery. In contrast, following brief optimization experiments, we found the Neon electroporator could impart unprecedented high transfection rates with GS cells. Further, the approach can be applied on both a small and large scale, allowing for cell-type specific optimization experiments.

SSCs are also similar to many other stem cell types in that they are rare and difficult to identify definitively through expression of particular proteins. Rather, rodent SSCs can be most strictly defined functionally based on their ability to home to a niche and colonize a recipient’s testes following transplantation, and then undergo meiosis and differentiate into sperm. Following years of intensive effort by multiple laboratories, conditions were eventually discovered for enriching for SSCs and maintaining them essentially indefinitely in vitro. The cultured cells, termed “germline stem cells”, have properties of untransformed primary cells that can be propagated long-term because of the self-renewal of SSCs. Importantly, putative SSCs can also be identified and cultured in vitro from human testes, although the duration for which human SSCs can be kept in vitro remains controversial and conditions for long term culture need to be SP600125 optimized. Given the robust nature of the rodent GS cell propagation system, we chose to model the process of ex vivo genome editing using mouse GS cells. We decided to test one of the more challenging genome editing approaches whereby homologous recombination is used to modify an existing mutation in the genome because this approach creates the precise modifications necessary for the most powerful research and therapy applications. For research purposes genome modification by homologous recombination greatly decreases the possibility of heterogeneous phenotypes from uncontrolled random integration; that is, with the latter, a transgene’s expression may be variable or silenced depending on where it integrates. For therapeutic purposes HR is potentially safer because of the elimination of random insertions, which in certain settings have been shown to lead to cancer through the process of insertional oncogenesis. While the frequency of HR with an exogenous DNA repair substrate in most cell types is too low to be therapeutically useful, the frequency can be increased by several orders of magnitude by introducing a double strand break at the site in the chromosome to be modified. Creation of a DSB can be accomplished using custom designed nucleases, including zinc finger nucleases, TAL effector nucleases or RNA guided endonucleases. ZFNs and TALENs are chimeric proteins comprising a nuclease domain from the type II restriction enzyme Fok I and a DNA binding domain engineered to recognize a specific sequence. ZFNs and TALENs have been demonstrated to stimulate homology directed repair of a DSB using an exogenous DNA repair substrate, or “gene targeting”, in a wide variety of contexts. For example, correction of a point mutation in interleukin 2 receptor, gamma was accomplished in K562 cells, an immortalized myelogenous leukemia human cell line, as well as in primary human T-cells and human CD34+ cells. Also, ZFNs and TALENs have been shown to simulate gene targeting in mouse and human embryonic stem cells and induced pluripotent stem cells. Still, genome engineering in the context of adult primary-like stem cells, which are likely to more closely resemble cells that will be used in therapy, is relatively unexplored. Moreover, the use of ZFN or TALEN stimulated HR to modify the genome in GS cells has not been described.

Such markers are advantageous because they facilitate the detection of functional variation and the signature of selection in genomic scans or association genetic studies. Transcriptbased SSRs are advantageous compared to SSRs in nontranscribed regions owing to their higher amplification rates and cross-species transferability. Currently, although many SSR markers were identified in the Fagaceae family, only a few SSR markers were reported in Q. pubescens. The predicted SSRs from the assembled transcriptome of Q. pubescens, will likely be of value for genetic analyses of Q. pubescens and other related nonmodel plants. In the recent years, transcriptome sequencing became a most powerful and efficient approach to uncover genomic information in non-model organism. The de novo assembly and annotation of the Q. pubescens transcriptome provided complete information concerning the expressed sequences of leaf tissue. Data of our study represent an important tool for discovering genes of interest and genetic markers, thus allowing investigation of the functional diversity in natural populations. Our characterization of the leaf transcriptome in Q. pubescens has not only enriched the publicly available database of sequences for members of the Quercus, but will also facilitate genetic analysis of other non-model organisms. Furthermore, our data demostrate that Illumina paired-end sequencing can successfully be applied as a rapid and cost-effective method to non-model organisms, especially those with large genomes and without prior genome annotation. Despite many research and sanitary efforts, tuberculosis remains one of the deadliest human infectious diseases far from being defeated. The poor knowledge of the biology of its causative agent, Mycobacterium tuberculosis, is a main obstacle toward the development of improved control strategies. In this context, a better understanding of surface exposed, secreted and cell wall associated proteins is classically a key step to dissect the mechanisms of pathogenesis of bacteria and to identify antigens that may serve as candidate vaccines. The complexity of the mycobacterial cell wall is such that only LY294002 recently it has been possible to solve its structure, including a peculiar outer membrane referred to as mycomembrane. Consequently, we still have limited knowledge regarding the proteins and protein apparatuses localizing in the mycomembrane and the molecular determinants mediating host-pathogen interactions. The recent discovery of the ESX secretion systems is shedding light on the mechanism whereby Mtb translocate effector proteins that are secreted or exposed on its surface and that can interfere with host components. The results of these studies are leading to the development of new vaccines and drug targets, emphasizing the impact that this line of research may have in the control of TB. Among the cell wall associated proteins are the PE_PGRSs, a family of around 60 proteins found only in members of the Mtb complex, in Mycobacterium ulcerans and Mycobacterium marinum. PE_PGRSs are characterized by a highly conserved PE domain, a central polymorphic PGRS domain and a unique Cterminal domain that may vary in size from few to up to 300 amino acids.

Altogether, the innervation role and the suggested involvement of IKAP in intracellular target derived signal transduction, specific gene expression together with cytoskeleton regulation in PNS neurons may explain in many ways the complexity of the FD phenotype that involves the selective loss of certain PNS neurons during development and after birth in FD patients. Early life experiences profoundly influence the later development, the structure and function of an organism.This phenomenon, called “developmental programming,” is a process whereby an environmental factor acting during a sensitive or vulnerable developmental period exerts effects that, in some cases, will persist throughout life. Adaptive or maladaptive responses to environmental stressors reflect an animal’s capacity to re-establish temporarily disrupted physiological homeostasis. A number of factors contribute to the qualitative nature of these responses such as: the intensity and duration of stressors, the individual’s ability to initiate an adaptive response, and the phase of the life when the stressor event occurs. In particular, concerning the latter point, during postnatal life, a critical period for neuroendocrinological and behavioural development processes, different emotional events may influence, in opposite ways, vulnerability to the effects of stress later in life, possibly by inducing a persistent sensitization in stressresponsive neural circuits. “Neonatal maternal deprivation” is one of the best known experimental animal models that well reproduces in rodents the consequence of traumatic experiences occurring in humans in early life. In particular, the stress evoked by altering mother–infant interactions during lactation causes the offspring, once adult, to develop a phenotype more susceptible to stress events and characterized by hyperactivation of the Hypothalamus – Pituitary – Ruxolitinib Adrenal axis. Interestingly, the pathophysiological modifications observed in adult rats affect not only the behaviour and the neuroendocrine system, but also the homeostasis of the gastrointestinal tract. In fact, adult rats separated early postnatally from their mothers have been found to be predisposed to colonic barrier dysfunction and to have an enhanced mucosal response to stress. These findings are in line with evidence that shows that adverse experiences early in life can have implications in the development and the clinical course of human intestinal disorders, including inflammatory bowel disease and intestinal bowel syndrome, where inflammatory and stress stimuli play primary roles. On the other hand, experiences, during human infancy, involving dynamic, tender, and stimulating environments, may have positive long lasting effects on the quality of life, can serve as a source of resilience in the face of chronic stress, and tend to promote resistance to stress and diminish vulnerability to stressinduced illness. In recent years, several experimental animal models have well represented this evidence. Environmental enrichment has been used as a procedure that might prevent some of the deleterious effects of stress.