Inclusion of genetically identical animals to a numerator relationship matrix and modification of its inverse

In the field of animal breeding, estimation of genetic parameters and prediction of breeding values are routinely conducted by analyzing quantitative traits. Using an animal model and including the direct inverse of a numerator relationship matrix (NRM) into a mixed model has made these analyses possible. However, a method including a genetically identical animal (GIA) in NRM if genetic relationships between pairs of GIAs are not perfect, is still lacking. Here, we describe a method to incorporate GIAs into NRM using a K matrix in which diagonal elements are set to 1.0, off-diagonal elements between pairs of GIAs to (1-x) and the other elements to 0, where x is a constant less than 0.05. The inverse of the K matrix is then calculated directly by a simple formula. Thus, the inverse of the NRM is calculated by the products of the lower triangular matrix that identifies the parents of each individual, its transpose matrix, the inverse of the K matrix and the inverse of diagonal matrix D, in which the diagonal elements comprise a number of known parents and their inbreeding coefficients. The computing method is adaptable to the analysis of a data set including pairs of GIAs with imperfect relationships

Cure of ADPKD by Selection for Spontaneous Genetic Repair Events in Pkd1-Mutated iPS Cells

Induced pluripotent stem cells (iPSCs) generated by epigenetic reprogramming of personal somatic cells have limited therapeutic capacity for patients suffering from genetic disorders. Here we demonstrate restoration of a genomic mutation heterozygous for Pkd1 (polycystic kidney disease 1) deletion (Pkd1(+/−) to Pkd1(+/R+)) by spontaneous mitotic recombination. Notably, recombination between homologous chromosomes occurred at a frequency of 1∼2 per 10,000 iPSCs. Southern blot hybridization and genomic PCR analyses demonstrated that the genotype of the mutation-restored iPSCs was indistinguishable from that of the wild-type cells. Importantly, the frequency of cyst generation in kidneys of adult chimeric mice containing Pkd1(+/R+) iPSCs was significantly lower than that of adult chimeric mice with parental Pkd1(+/−) iPSCs, and indistinguishable from that of wild-type mice. This repair step could be directly incorporated into iPSC development programmes prior to cell transplantation, offering an invaluable step forward for patients carrying a wide range of genetic disorders

Identification of Inappropriately Reprogrammed Genes by Large-Scale Transcriptome Analysis of Individual Cloned Mouse Blastocysts

Although cloned embryos generated by somatic/embryonic stem cell nuclear transfer (SECNT) certainly give rise to viable individuals, they can often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. In an effort to gain further insights into reprogramming and the properties of SECNT embryos, we performed a large-scale gene expression profiling of 87 single blastocysts using GeneChip microarrays. Sertoli cells, cumulus cells, and embryonic stem cells were used as donor cells. The gene expression profiles of 87 blastocysts were subjected to microarray analysis. Using principal component analysis and hierarchical clustering, the gene expression profiles were clearly classified into 3 clusters corresponding to the type of donor cell. The results revealed that each type of SECNT embryo had a unique gene expression profile that was strictly dependent upon the type of donor cells, although there was considerable variation among the individual profiles within each group. This suggests that the reprogramming process is distinct for embryos cloned from different types of donor cells. Furthermore, on the basis of the results of comparison analysis, we identified 35 genes that were inappropriately reprogrammed in most of the SECNT embryos; our findings demonstrated that some of these genes, such as Asz1, Xlr3a and App, were appropriately reprogrammed only in the embryos with a transcriptional profile that was the closest to that of the controls. Our findings provide a framework to further understand the reprogramming in SECNT embryos

Characterization of Spermatogonial Stem Cells Lacking Intercellular Bridges and Genetic Replacement of a Mutation in Spermatogonial Stem Cells

Stem cells have a potential of gene therapy for regenerative medicine. Among various stem cells, spermatogonial stem cells have a unique characteristic in which neighboring cells can be connected by intercellular bridges. However, the roles of intercellular bridges for stem cell self-renewal, differentiation, and proliferation remain to be elucidated. Here, we show not only the characteristics of testis-expressed gene 14 (TEX14) null spermatogonial stem cells lacking intercellular bridges but also a trial application of genetic correction of a mutation in spermatogonial stem cells as a model for future gene therapy. In TEX14 null testes, some genes important for undifferentiated spermatogonia as well as some differentiation-related genes were activated. TEX14 null spermatogonial stem cells, surprisingly, could form chain-like structures even though they do not form stable intercellular bridges. TEX14 null spermatogonial stem cells in culture possessed both characteristics of undifferentiated and differentiated spermatogonia. Long-term culture of TEX14 null spermatogonial stem cells could not be established likely secondary to up-regulation of CDK4 inhibitors and down-regulation of cyclin E. These results suggest that intercellular bridges are essential for both maintenance of spermatogonial stem cells and their proliferation. Lastly, a mutation in Tex14+/− spermatogonial stem cells was successfully replaced by homologous recombination in vitro. Our study provides a therapeutic potential of spermatogonial stem cells for reproductive medicine if they can be cultured long-term

Chemically induced DNA hypomethylation in breast carcinoma cells detected by the amplification of intermethylated sites

INTRODUCTION: Compromised patterns of gene expression result in genomic instability, altered patterns of gene expression and tumour formation. Specifically, aberrant DNA hypermethylation in gene promoter regions leads to gene silencing, whereas global hypomethylation events can result in chromosomal instability and oncogene activation. Potential links exist between environmental agents and DNA methylation, but the destabilizing effects of environmental exposures on the DNA methylation machinery are not understood within the context of breast cancer aetiology. METHODS: We assessed genome-wide changes in methylation patterns using a unique methylation profiling technique called amplification of intermethylated sites (AIMS). This method generates easily readable fingerprints that represent the investigated cell line's methylation profile, based on the differential cleavage of DNA with methylation-specific isoschisomeric restriction endonucleases. RESULTS: We validated this approach by demonstrating both unique and reoccurring sites of genomic hypomethylation in four breast carcinoma cell lines treated with the cytosine analogue 5-azacytidine. Comparison of treated with control samples revealed individual bands that exhibited methylation changes, and these bands were excized and cloned, and the precise genomic location individually identified. In most cases, these regions of hypomethylation coincided with susceptible target regions previously associated with chromosome breakage, rearrangement and gene amplification. Similarly, we observed that acute benzopyrene exposure is associated with altered methylation patterns in these cell lines. CONCLUSION: These results reinforce the link between environmental exposures, DNA methylation and breast cancer, and support a role for AIMS as a rapid, affordable screening method to identify environmentally induced DNA methylation changes that occur in tumourigenesis

Birthweight, Maternal Weight Trajectories and Global DNA Methylation of LINE-1 Repetitive Elements

Low birthweight, premature birth, intrauterine growth retardation, and maternal malnutrition have been related to an increased risk of cardiovascular disease, type 2 diabetes mellitus, obesity, and neuropsychiatric disorders later in life. Conversely, high birthweight has been linked to future risk of cancer. Global DNA methylation estimated by the methylation of repetitive sequences in the genome is an indicator of susceptibility to chronic diseases. We used data and biospecimens from an epigenetic birth cohort to explore the association between trajectories of fetal and maternal weight and LINE-1 methylation in 319 mother-child dyads. Newborns with low or high birthweight had significantly lower LINE-1 methylation levels in their cord blood compared to normal weight infants after adjusting for gestational age, sex of the child, maternal age at delivery, and maternal smoking during pregnancy (p = 0.007 and p = 0.036, respectively), but the magnitude of the difference was small. Infants born prematurely also had lower LINE-1 methylation levels in cord blood compared to term infants, and this difference, though small, was statistically significant (p = 0.004). We did not find important associations between maternal prepregnancy BMI or gestational weight gain and global methylation of the cord blood or fetal placental tissue. In conclusion, we found significant differences in cord blood LINE-1 methylation among newborns with low and high birthweight as well as among prematurely born infants. Future studies may elucidate whether chromosomal instabilities or other functional consequences of these changes contribute to the increased risk of chronic diseases among individuals with these characteristics

Optimizing the Design of Oligonucleotides for Homology Directed Gene Targeting

BACKGROUND: Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit. METHODOLOGY/PRINCIPAL FINDINGS: In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA) polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex. CONCLUSION AND SIGNIFICANCE: A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases) in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA

Parkinson's Disease DJ-1 L166P Alters rRNA Biogenesis by Exclusion of TTRAP from the Nucleolus and Sequestration into Cytoplasmic Aggregates via TRAF6

Mutations in PARK7/DJ-1 gene are associated to autosomal recessive early onset forms of Parkinson's disease (PD). Although large gene deletions have been linked to a loss-of-function phenotype, the pathogenic mechanism of missense mutations is less clear. The L166P mutation causes misfolding of DJ-1 protein and its degradation. L166P protein may also accumulate into insoluble cytoplasmic aggregates with a mechanism facilitated by the E3 ligase TNF receptor associated factor 6 (TRAF6). Upon proteasome impairment L166P activates the JNK/p38 MAPK apoptotic pathway by its interaction with TRAF and TNF Receptor Associated Protein (TTRAP). When proteasome activity is blocked in the presence of wild-type DJ-1, TTRAP forms aggregates that are localized to the cytoplasm or associated to nucleolar cavities, where it is required for a correct rRNA biogenesis. In this study we show that in post-mortem brains of sporadic PD patients TTRAP is associated to the nucleolus and to Lewy Bodies, cytoplasmic aggregates considered the hallmark of the disease. In SH-SY5Y neuroblastoma cells, misfolded mutant DJ-1 L166P alters rRNA biogenesis inhibiting TTRAP localization to the nucleolus and enhancing its recruitment into cytoplasmic aggregates with a mechanism that depends in part on TRAF6 activity. This work suggests that TTRAP plays a role in the molecular mechanisms of both sporadic and familial PD. Furthermore, it unveils the existence of an interplay between cytoplasmic and nucleolar aggregates that impacts rRNA biogenesis and involves TRAF6

dSETDB1 and SU(VAR)3–9 Sequentially Function during Germline-Stem Cell Differentiation in Drosophila melanogaster

Germline-stem cells (GSCs) produce gametes and are thus true “immortal stem cells”. In Drosophila ovaries, GSCs divide asymmetrically to produce daughter GSCs and cystoblasts, and the latter differentiate into germline cysts. Here we show that the histone-lysine methyltransferase dSETDB1, located in pericentric heterochromatin, catalyzes H3-K9 trimethylation in GSCs and their immediate descendants. As germline cysts differentiate into egg chambers, the dSETDB1 function is gradually taken over by another H3-K9-specific methyltransferase, SU(VAR)3–9. Loss-of-function mutations in dsetdb1 or Su(var)3–9 abolish both H3K9me3 and heterochromatin protein-1 (HP1) signals from the anterior germarium and the developing egg chambers, respectively, and cause localization of H3K9me3 away from DNA-dense regions in most posterior germarium cells. These results indicate that dSETDB1 and SU(VAR)3–9 act together with distinct roles during oogenesis, with dsetdb1 being of particular importance due to its GSC-specific function and more severe mutant phenotype