Establishment of regeneration and transformation protocols to create hypoalleregenic peanut (Arachis hypogaea) and mustard (Brassica juncea) through genome editing

A stable and reproducible in vitro regeneration system is necessary for genetic engineering, however, explant responses vary widely between and within species and are highly dependent on the culture conditions. Despite decades of research, plant regeneration is still challenging especially with some plant species referred to as recalcitrant. In this study, we investigated the regeneration and genetic transformation capacities of different lines of peanut (Arachis hypogaea) that is considered recalcitrant and Brown mustard (Brassica juncea) in order to enable editing of the allergens Ara h 1 and Bra J I in peanut and mustard, respectively, via CRISPR/Cas9. Moreover, we demonstrated that, several of the first edited mustard lines displayed reduced or total absence of the Bra J I protein. Firstly, the factors affecting the adventitious shoot regeneration of both plant species were investigated using four peanut lines and six mustard lines from two geographical regions (Europe and India). In both species, shoot regeneration was significantly influenced by the explant type and the genetic make-up of the different lines tested. In mustard, the 5 days old cotyledon explants of all lines showed better responses than hypocotyls regarding adventitious shoot regeneration whereas in peanut the leaflet explants of 5-day-old seedlings exhibited superiority. The combinations of different types of cytokinins and auxins were tested on the explants of both plant species. In all peanut lines, the leaflet explants responded best on medium with 22.19 µM 6-benzylaminopurine (BAP) (+ 2.3 µM kinetin), which was reduced to 7.40 µM BAP after eight weeks, with regeneration rates of 10-89.1% and a mean shoot number per regenerating explant of 1-3.1 shoots. For mustard, the medium containing 8.88 µM BAP, 5.37 µM 1-naphthaleneacetic acid (NAA), and 9.95 µM Silver nitrate (AgNO3) resulted in the highest shoot regeneration rates (58-72% and 65-90% for the European and Indian lines, respectively) as well as the highest shoot numbers per regenerating explant (2.2-2.7 and 2.3-3.0). Furthermore, the effect of different light qualities on shoot regeneration from leaflet explants of the peanut lines was investigated in order to promote shoot induction and elongation. A strong effect of the culture temperature on the regeneration efficiency was observed as different light treatments were connected with different culture temperatures. However, red and blue LEDs could substitute tubular fluorescent lamps without affecting shoot regeneration. In the second step, Agrobacterium-mediated transformation of both plant species was investigated. Transgenic plants carrying large deletions of 566 up to 790 bp as well as indels in the targeted regions especially, indels in all four Bra j I alleles were obtained with the mustard lines tested. Seed viability was investigated in several transgenic mustard lines through in vitro and ex vitro germination. A decrease in seed viability and seed formation was observed in some edited lines, which indicated that the mutation of the major allergen Bra J I in mustard affected seed development. Part of the seeds exhibited aberrant phenotypes that resulted in the rupture of the testa already in the siliques. In contrast, the regenerated shoots from the different transformation experiments with various peanut lines exhibited a lack of transgenicity

Transcriptome Analysis during Human Trophectoderm Specification Suggests New Roles of Metabolic and Epigenetic Genes

In humans, successful pregnancy depends on a cascade of dynamic events during early embryonic development. Unfortunately, molecular data on these critical events is scarce. To improve our understanding of the molecular mechanisms that govern the specification/development of the trophoblast cell lineage, the transcriptome of human trophectoderm (TE) cells from day 5 blastocysts was compared to that of single day 3 embryos from our in vitro fertilization program by using Human Genome U133 Plus 2.0 microarrays. Some of the microarray data were validated by quantitative RT-PCR. The TE molecular signature included 2,196 transcripts, among which were genes already known to be TE-specific (GATA2, GATA3 and GCM1) but also genes involved in trophoblast invasion (MUC15), chromatin remodeling (specifically the DNA methyltransferase DNMT3L) and steroid metabolism (HSD3B1, HSD17B1 and FDX1). In day 3 human embryos 1,714 transcripts were specifically up-regulated. Besides stemness genes such as NANOG and DPPA2, this signature included genes belonging to the NLR family (NALP4, 5, 9, 11 and 13), Ret finger protein-like family (RFPL1, 2 and 3), Melanoma Antigen family (MAGEA1, 2, 3, 5, 6 and 12) and previously unreported transcripts, such as MBD3L2 and ZSCAN4. This study provides a comprehensive outlook of the genes that are expressed during the initial embryo-trophectoderm transition in humans. Further understanding of the biological functions of the key genes involved in steroidogenesis and epigenetic regulation of transcription that are up-regulated in TE cells may clarify their contribution to TE specification and might also provide new biomarkers for the selection of viable and competent blastocysts

Dissecting the First Transcriptional Divergence During Human Embryonic Development

The trophoblast cell lineage is specified early at the blastocyst stage, leading to the emergence of the trophectoderm and the pluripotent cells of the inner cell mass. Using a double mRNA amplification technique and a comparison with transcriptome data on pluripotent stem cells, placenta, germinal and adult tissues, we report here some essential molecular features of the human mural trophectoderm. In addition to genes known for their role in placenta (CGA, PGF, ALPPL2 and ABCG2), human trophectoderm also strongly expressed Laminins, such as LAMA1, and the GAGE Cancer/Testis genes. The very high level of ABCG2 expression in trophectoderm, 7.9-fold higher than in placenta, suggests a major role of this gene in shielding the very early embryo from xenobiotics. Several genes, including CCKBR and DNMT3L, were specifically up-regulated only in trophectoderm, indicating that the trophoblast cell lineage shares with the germinal lineage a transient burst of DNMT3L expression. A trophectoderm core transcriptional regulatory circuitry formed by 13 tightly interconnected transcription factors (CEBPA, GATA2, GATA3, GCM1, KLF5, MAFK, MSX2, MXD1, PPARD, PPARG, PPP1R13L, TFAP2C and TP63), was found to be induced in trophectoderm and maintained in placenta. The induction of this network could be recapitulated in an in vitro trophoblast differentiation model

StemCellNet: an interactive platform for network-oriented investigations in stem cell biology.

Stem cells are characterized by their potential for self-renewal and their capacity to differentiate into mature cells. These two key features emerge through the interplay of various factors within complex molecular networks. To provide researchers with a dedicated tool to investigate these networks, we have developed StemCellNet, a versatile web server for interactive network analysis and visualization. It rapidly generates focused networks based on a large collection of physical and regulatory interactions identified in human and murine stem cells. The StemCellNet web-interface has various easy-to-use tools for selection and prioritization of network components, as well as for integration of expression data provided by the user. As a unique feature, the networks generated can be screened against a compendium of stemness-associated genes. StemCellNet can also indicate novel candidate genes by evaluating their connectivity patterns. Finally, an optional dataset of generic interactions, which provides large coverage of the human and mouse proteome, extends the versatility of StemCellNet to other biomedical research areas in which stem cells play important roles, such as in degenerative diseases or cancer. The StemCellNet web server is freely accessible at http://stemcellnet.sysbiolab.eu

Transcriptome Profiling of Human Pre-Implantation Development

BACKGROUND: Preimplantation development is a crucial step in early human development. However, the molecular basis of human preimplantation development is not well known. METHODOLOGY: By applying microarray on 397 human oocytes and embryos at six developmental stages, we studied the transcription dynamics during human preimplantation development. PRINCIPAL FINDINGS: We found that the preimplantation development consisted of two main transitions: from metaphase-II oocyte to 4-cell embryo where mainly the maternal genes were expressed, and from 8-cell embryo to blastocyst with down-regulation of the maternal genes and up-regulation of embryonic genes. Human preimplantation development proved relatively autonomous. Genes predominantly expressed in oocytes and embryos are well conserved during evolution. SIGNIFICANCE: Our database and findings provide fundamental resources for understandin

Characterising and Predicting Haploinsufficiency in the Human Genome

Haploinsufficiency, wherein a single functional copy of a gene is insufficient to maintain normal function, is a major cause of dominant disease. Human disease studies have identified several hundred haploinsufficient (HI) genes. We have compiled a map of 1,079 haplosufficient (HS) genes by systematic identification of genes unambiguously and repeatedly compromised by copy number variation among 8,458 apparently healthy individuals and contrasted the genomic, evolutionary, functional, and network properties between these HS genes and known HI genes. We found that HI genes are typically longer and have more conserved coding sequences and promoters than HS genes. HI genes exhibit higher levels of expression during early development and greater tissue specificity. Moreover, within a probabilistic human functional interaction network HI genes have more interaction partners and greater network proximity to other known HI genes. We built a predictive model on the basis of these differences and annotated 12,443 genes with their predicted probability of being haploinsufficient. We validated these predictions of haploinsufficiency by demonstrating that genes with a high predicted probability of exhibiting haploinsufficiency are enriched among genes implicated in human dominant diseases and among genes causing abnormal phenotypes in heterozygous knockout mice. We have transformed these gene-based haploinsufficiency predictions into haploinsufficiency scores for genic deletions, which we demonstrate to better discriminate between pathogenic and benign deletions than consideration of the deletion size or numbers of genes deleted. These robust predictions of haploinsufficiency support clinical interpretation of novel loss-of-function variants and prioritization of variants and genes for follow-up studies

Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells

The identification of stem cells within a mixed population of cells is a major hurdle for stem cell biology–in particular, in the identification of induced pluripotent stem (iPS) cells during the reprogramming process. Based on the selective expression of stem cell surface markers, a method to specifically infect stem cells through antibody-conjugated lentiviral particles has been developed that can deliver both visual markers for live-cell imaging as well as selectable markers to enrich for iPS cells. Antibodies recognizing SSEA4 and CD24 mediated the selective infection of the iPS cells over the parental human fibroblasts, allowing for rapid expansion of these cells by puromycin selection. Adaptation of the vector allows for the selective marking of human embryonic stem (hES) cells for their removal from a population of differentiated cells. This method has the benefit that it not only identifies stem cells, but that specific genes, including positive and negative selection markers, regulatory genes or miRNA can be delivered to the targeted stem cells. The ability to specifically target gene delivery to human pluripotent stem cells has broad applications in tissue engineering and stem cell therapies

Methods for comprehensive chromosome screening of oocytes and embryos: capabilities, limitations, and evidence of validity

Preimplantation aneuploidy screening of cleavage stage embryos using fluorescence in situ hybridization (FISH) may no longer be considered the standard of care in reproductive medicine. Over the last few years, there has been considerable development of novel technologies for comprehensive chromosome screening (CCS) of the human genome. Among the notable methodologies that have been incorporated are whole genome amplification, metaphase and array based comparative genomic hybridization, single nucleotide polymorphism microarrays, and quantitative real-time PCR. As these methods become more integral to treating patients with infertility, it is critical that clinicians and scientists obtain a better understanding of their capabilities and limitations. This article will focus on reviewing these technologies and the evidence of their validity

An embryonic stem cell–like gene expression signature in poorly differentiated aggressive human tumors

Cancer cells possess traits reminiscent of those ascribed to normal stem cells. It is unclear, however, whether these phenotypic similarities reflect the activity of common molecular pathways. Here, we analyze the enrichment patterns of gene sets associated with embryonic stem (ES) cell identity in the expression profiles of various human tumor types. We find that histologically poorly differentiated tumors show preferential overexpression of genes normally enriched in ES cells, combined with preferential repression of Polycomb-regulated genes. Moreover, activation targets of Nanog, Oct4, Sox2 and c-Myc are more frequently overexpressed in poorly differentiated tumors than in well-differentiated tumors. In breast cancers, this ES-like signature is associated with high-grade estrogen receptor (ER)-negative tumors, often of the basal-like subtype, and with poor clinical outcome. The ES signature is also present in poorly differentiated glioblastomas and bladder carcinomas. We identify a subset of ES cell-associated transcription regulators that are highly expressed in poorly differentiated tumors. Our results reveal a previously unknown link between genes associated with ES cell identity and the histopathological traits of tumors and support the possibility that these genes contribute to stem cell–like phenotypes shown by many tumors

Empirical comparison of cross-platform normalization methods for gene expression data

<p>Abstract</p> <p>Background</p> <p>Simultaneous measurement of gene expression on a genomic scale can be accomplished using microarray technology or by sequencing based methods. Researchers who perform high throughput gene expression assays often deposit their data in public databases, but heterogeneity of measurement platforms leads to challenges for the combination and comparison of data sets. Researchers wishing to perform cross platform normalization face two major obstacles. First, a choice must be made about which method or methods to employ. Nine are currently available, and no rigorous comparison exists. Second, software for the selected method must be obtained and incorporated into a data analysis workflow.</p> <p>Results</p> <p>Using two publicly available cross-platform testing data sets, cross-platform normalization methods are compared based on inter-platform concordance and on the consistency of gene lists obtained with transformed data. Scatter and ROC-like plots are produced and new statistics based on those plots are introduced to measure the effectiveness of each method. Bootstrapping is employed to obtain distributions for those statistics. The consistency of platform effects across studies is explored theoretically and with respect to the testing data sets.</p> <p>Conclusions</p> <p>Our comparisons indicate that four methods, DWD, EB, GQ, and XPN, are generally effective, while the remaining methods do not adequately correct for platform effects. Of the four successful methods, XPN generally shows the highest inter-platform concordance when treatment groups are equally sized, while DWD is most robust to differently sized treatment groups and consistently shows the smallest loss in gene detection. We provide an R package, CONOR, capable of performing the nine cross-platform normalization methods considered. The package can be downloaded at <url>http://alborz.sdsu.edu/conor</url> and is available from CRAN.</p