Hydractinia is a colonial marine hydroid that exhibits remarkable biological properties, including the capacity to regenerate its entire body throughout its lifetime, a process made possible by its adult migratory stem cells, known as i-cells. Here, we provide an in-depth characterization of the genomic structure and gene content of two Hydractinia species, H. symbiolongicarpus and H. echinata, placing them in a comparative evolutionary framework with other cnidarian genomes. We also generated and annotated a single-cell transcriptomic atlas for adult male H. symbiolongicarpus and identified cell type markers for all major cell types, including key i-cell markers. Orthology analyses based on the markers revealed that Hydractinia's i-cells are highly enriched in genes that are widely shared amongst animals, a striking finding given that Hydractinia has a higher proportion of phylum-specific genes than any of the other 41 animals in our orthology analysis. These results indicate that Hydractinia's stem cells and early progenitor cells may use a toolkit shared with all animals, making it a promising model organism for future exploration of stem cell biology and regenerative medicine. The genomic and transcriptomic resources for Hydractinia presented here will enable further studies of their regenerative capacity, colonial morphology, and ability to distinguish self from non-self.

Most colonial marine invertebrates are capable of allorecognition, the ability to distinguish between themselves and conspecifics. One long-standing question is whether invertebrate allorecognition genes are homologous to vertebrate histocompatibility genes. In the cnidarian Hydractinia symbiolongicarpus, allorecognition is controlled by at least two genes, Allorecognition 1 ( Alr1 ) and Allorecognition 2 ( Alr2 ), which encode highly polymorphic cell-surface proteins that serve as markers of self. Here, we show that Alr1 and Alr2 are part of a family of 41 Alr genes, all of which reside in a single genomic interval called the Allorecognition Complex (ARC). Using sensitive homology searches and highly accurate structural predictions, we demonstrate that the Alr proteins are members of the immunoglobulin superfamily (IgSF) with V-set and I-set Ig domains unlike any previously identified in animals. Specifically, their primary amino acid sequences lack many of the motifs considered diagnostic for V-set and I-set domains, yet they adopt secondary and tertiary structures nearly identical to canonical Ig domains. Thus, the V-set domain, which played a central role in the evolution of vertebrate adaptive immunity, was present in the last common ancestor of cnidarians and bilaterians. Unexpectedly, several Alr proteins also have immunoreceptor tyrosine-based activation motifs and immunoreceptor tyrosine-based inhibitory motifs in their cytoplasmic tails, suggesting they could participate in pathways homologous to those that regulate immunity in humans and flies. This work expands our definition of the IgSF with the addition of a family of unusual members, several of which play a role in invertebrate histocompatibility.

Invertebrate histocompatibility—also known as allorecognition—has long interested marine ecologists, population geneticists, evolutionary biologists, and immunologists, but its genetic basis remains enigmatic in most species. Here, we report the nearly complete sequence of a histocompatibility complex from the colonial cnidarian, Hydractinia symbiolongicarpus. This sequence reveals that the two known Hydractinia allorecognition genes, Allorecognition 1 (Alr1) and Allorecognition 2 (Alr2) are part of a large family of immunoglobulin superfamily (IgSF) genes, several of which are candidates for new allodeterminants. These genes encode transmembrane proteins with domain architectures similar to cell-adhesion molecules and immune receptors. Several also contain cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs) and immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Our data, which include highly accurate protein structure predictions, reveal that these proteins have V-set and I-set domains with unusual sequence signatures. This suggests the last common ancestor of cnidarians and bilaterians had distinct V-set and I-set Ig domains.

To address the void in the availability of high-quality proteomic data traversing the animal tree, we have implemented a pipeline for generating de novo assemblies based on publicly available data from the NCBI Sequence Read Archive, yielding a comprehensive collection of proteomes from 100 species spanning 21 animal phyla. We have also created the Animal Proteome Database (AniProtDB), a resource providing open access to this collection of high-quality metazoan proteomes, along with information on predicted proteins and protein domains for each taxonomic classification and the ability to perform sequence similarity searches against all proteomes generated using this pipeline. This solution vastly increases the utility of these data by removing the barrier to access for research groups who do not have the expertise or resources to generate these data themselves and enables the use of data from nontraditional research organisms that have the potential to address key questions in biomedicine.

Following the completion of the genome sequencing and gene prediction of Mnemiopsis leidyi, a lobate ctenophore that is native to the coastal waters of the western Atlantic Ocean, we developed and implemented the Mnemiopsis Genome Project Portal (MGP Portal), a comprehensive Web-based data portal for navigating the genome sequence and gene annotations. In the years following the first release of the MGP Portal, it has become evident that the inclusion of data from significant published studies on Mnemiopsis has been critical to its adoption as the centralized resource for this emerging model organism. With this most recent update, the Portal has significantly expanded to include in situ images, temporal developmental expression profiles and single-cell expression data. Recent enhancements also include implementations of an updated BLAST interface, new graphical visualization tools and updates to gene pages that integrate all new data types. Database URL: https://research.nhgri.nih.gov/mnemiopsis/.

How individuals perceive uncertainties in sequencing results may affect their clinical utility. The purpose of this study was to explore perceptions of uncertainties in carrier results and how they relate to psychological well-being and health behavior. Post-reproductive adults (N = 462) were randomized to receive carrier results from sequencing through either a web platform or a genetic counselor. On average, participants received two results. Group differences in affective, evaluative, and clinical uncertainties were assessed from baseline to 1 and 6 months; associations with test-specific distress and communication of results were assessed at 6 months. Reductions in affective uncertainty (∆x̅ = 0.78, 95% CI: 0.53, 1.02) and evaluative uncertainty (∆x̅ = 0.69, 95% CI: 0.51, 0.87) followed receipt of results regardless of randomization arm at 1 month. Participants in the web platform arm reported greater clinical uncertainty than those in the genetic counselor arm at 1 and 6 months; this was corroborated by the 1,230 questions asked of the genetic counselor and residual questions reported by those randomized to the web platform. Evaluative uncertainty was associated with a lower likelihood of communicating results to health care providers. Clinical uncertainty was associated with a lower likelihood of communicating results to children. Learning one’s carrier results may reduce perceptions of uncertainties, though web-based return may lead to less reduction in clinical uncertainty in the short term. These findings warrant reinforcement of clinical implications to minimize residual questions and promote appropriate health behavior (communicating results to at-risk relatives in the case of carrier results), especially when testing alternative delivery models.

Adeno-associated viral (AAV) vectors have emerged as the preferred platform for in vivo gene transfer because of their combined efficacy and safety. However, insertional mutagenesis with the subsequent development of hepatocellular carcinomas (HCCs) has been recurrently noted in newborn mice treated with high doses of AAV, and more recently, the association of wild-type AAV integrations in a subset of human HCCs has been documented. Here, we address, in a comprehensive, prospective study, the long-term risk of tumorigenicity in young adult mice following delivery of single-stranded AAVs targeting liver. HCC incidence in mice treated with therapeutic and reporter AAVs was low, in contrast to what has been previously documented in mice treated as newborns with higher doses of AAV. Specifically, HCCs developed in 6 out 76 of AAV-treated mice, and a pathogenic integration of AAV was found in only one tumor. Also, no evidence of liver tumorigenesis was found in juvenile AAV-treated mucopolysaccharidosis type VI (MPS VI) cats followed as long as 8 years after vector administration. Together, our results support the low risk of tumorigenesis associated with AAV-mediated gene transfer targeting juvenile/young adult livers, although constant monitoring of subjects enrolled in AAV clinical trial is advisable.

New sequencing technologies are becoming increasingly available in a variety of contexts. These techniques include whole-exome (we refer to this as “exome” sequencing) and whole-genome sequencing (Biesecker and Green 2014). These types of genomic sequencing have impacted both research and clinical practice. Genomic sequencing has led to the discovery of novel genetic etiologies of disease and has also improved the ability to diagnose patients with subtle or atypical presentations of genetic conditions (especially those affected by relatively rare disorders) by allowing simultaneous interrogation of many loci (Boycott et al. 2013; Yang et al. 2013, 2014; Taylor et al. 2015). To quantify genetic progress and the impact of these technologies in understanding the causes of Mendelian disorders, we analyzed methods of discovery in the last ~2.5 years. We reviewed all newly described Mendelian disease genes in the ~2.5-year period (April 30, 2013–November 30, 2015) following the initial public dissemination of the Clinical Genomic Database (CGD) (Solomon et al. 2013), a freely available web-based resource that focuses on the clinical sequelae and management of genetic disorders (the CGD, which is regularly updated to keep pace with genetic knowledge, is available at: http://research.nhgri.nih.gov/CGD/). Regarding relevant literature, as there is frequently a gap between manuscript acceptance, electronic, and final publication, we attempted to include only those genes/conditions in the ~2.5 year period that were published (available in PubMed) and subsequently included in both the CGD and Online Mendelian Inheritance in Man (OMIM, available at http://www.omim.org) within that ~2.5 year interval. We recognize this is imperfect for several reasons. These reasons include the lag between publication and incorporation in these databases, and the fact that the databases do not completely capture the literature and are themselves being continually being refined and updated. However, with these caveats in terms of potential inaccuracies, the trends that we reveal are overall interesting. For each article, we determined how the discovery was made (e.g., through whole-genome sequencing alone, homozygosity mapping and exome sequencing, candidate gene studies, or a combination of possibilities). If the discovery involved findings through a previous publication on the same families or condition (such as linkage analysis implicating specific loci), we included that previous method as part of the discovery process. In the example given, this would be considered “exome + linkage.” We also determined if the discovery was made through analysis of a single individual; a single family; or a single individual or family followed by the identification of additional mutation-positive individuals through studies of a larger cohort. Finally, we investigated whether additional bench-based studies, aimed at providing understanding and evidence beyond clinical and bioinformatics results, were included in the investigation. We did not include newly reported conditions allelic to previously described conditions. Regarding this latter exclusion criterion, though many genes have clinically distinct allelic disorders, we wished to be maximally conservative in order to avoid any controversy arising from similar disorders that may represent the spectrum of a single disease entity. For newly discovered genetic causes, 445 new genes were identified in the 2.5-year period in the defined intervals (see Fig. 1 and Table S1 for details). A number of genes were identified by apparently independent studies with separate but simultaneous publications. Methods from each such independent study were counted separately here, such that 492 individual studies are represented. Of the 492 studies, 394 (80%) used some type of genomic sequencing (including exome or genome sequencing but not X-chromosome exome or mitochondrial exome). Three hundred and seventy-nine (77%) used exome sequencing, 11 (2%) used genome sequencing, and four (1%) used both exome and genome sequencing in the same study. Two hundred and thirty-one (47%) used only exome sequencing (without other investigations such as homozygosity mapping) for gene identification; 238 (48%) used only exome and/or genome sequencing. Another 156 (32%) used exome or genome sequencing in addition to other techniques, such as homozygosity mapping. Forty-one (8%) used a traditional candidate gene approach without other methods, though 46 (10%) used a candidate gene approach with methods other than exome or genome sequencing. The remaining 11 (2%) used other methods, such as cytogenomic methods alone, X-chromosome exome sequencing, or mitochondrial sequencing (see Table S1). Eighteen (4%) of the overall studies investigated a single patient only; 114 (23%) studied multiple members of a single family; 93 (19%) were initially done on a single patient or family but then investigated a larger cohort based on these initial findings and identified and reported additional mutation-positive individuals. The remaining 267 (54%) were reported as studying multiple patients or families simultaneously. We would predict that over time, the use of genomic sequencing methods will eclipse other methods of identifying disease genes. To detect if there has been a shift in methods, even over the short ~2.5 year period of this study, we sorted the 492 publications by PubMed identification number (PMID) and split the publications into two groups. We recognize that the PMID identifiers do not precisely capture chronology. Nevertheless, we note that studies in the second group, which represents manuscripts published more recently, made significantly more use of genomic sequencing overall (210 vs. 184 studies, P = 0.0046 by Fisher's exact test) and genomic sequencing alone (135 vs. 103 studies, P = 0.0051 by Fisher's exact test) than did those in the first. Of the 492 studies, 359 (73%) included some type of laboratory-based assay in addition to standard clinical work-up and genomic sequencing and analysis. However, we intentionally did not further analyze the type of cellular or functional analyses that were done as part of the paper – we viewed this assignment as difficult and potentially unhelpful to interpret, as this depended on the availability of previous knowledge about a particular gene's function (e.g., through an animal model), the specific mutation in question (e.g., a truncating vs. missense mutation), may have been aimed at understanding the biological implications of the genetic pathways involved rather than or in addition to the question of mutation pathogenicity, and because so many different possible methods might have been used (MacArthur et al. 2014). Our analyses show that exome sequencing currently accounts for the vast majority of causal gene discovery. Almost half of all the discoveries investigated here occurred through exome sequencing alone. This is anticipated to shift to genome sequencing as affordability and accuracy of sequencing continue to improve (Hayden 2014). Additionally, our analyses show a statistically significant increase in the use of exome or genome sequencing even within the short time period analyzed. Again, though we admit to potential inaccuracies as the background databases shift, we feel that the trends described here are illustrative. In conclusion, new sequencing technologies are resulting in a dramatic change in the discovery of the causes of disease. These discoveries can be quickly translated into clinical care – patients affected with many conditions now have a better chance of explanations based on genetic testing. Further, finding the molecular etiology of a condition may then in turn yield more tailored and overall better management (Solomon et al. 2013; Soden et al. 2014; Khromykh and Solomon 2015; Solomon 2015; Willig et al. 2015). It will be interesting to perform similar analyses in the future as new techniques emerge and as the body of knowledge of the causes of human disease continues to grow and evolve. This research was supported in part by the Intramural Research Program of the National Human Genome Research Institute, the National Institutes of Health. None of the authors have disclosures or conflicts of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

, (2013); 342 Science et al. Joseph F. Ryan Implications for Cell Type Evolution and Its Mnemiopsis leidyi The Genome of the Ctenophore This copy is for your personal, non-commercial use only. clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ): November 10, 2014 www.sciencemag.org (this information is current as of The following resources related to this article are available online at http://www.sciencemag.org/content/342/6164/1242592.full.html version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/content/suppl/2013/12/11/342.6164.1242592.DC1.html can be found at: Supporting Online Material http://www.sciencemag.org/content/342/6164/1242592.full.html#related found at: can be related to this article A list of selected additional articles on the Science Web sites http://www.sciencemag.org/content/342/6164/1242592.full.html#ref-list-1 , 13 of which can be accessed free: cites 49 articles This article http://www.sciencemag.org/content/342/6164/1242592.full.html#related-urls 16 articles hosted by HighWire Press; see: cited by This article has been http://www.sciencemag.org/cgi/collection/evolution Evolution subject collections: This article appears in the following

Background Mnemiopsis leidyi is a ctenophore native to the coastal waters of the western Atlantic Ocean. A number of studies on Mnemiopsis have led to a better understanding of many key biological processes, and these studies have contributed to the emergence of Mnemiopsis as an important model for evolutionary and developmental studies. Recently, we sequenced, assembled, annotated, and performed a preliminary analysis on the 150-megabase genome of the ctenophore, Mnemiopsis . This sequencing effort has produced the first set of whole-genome sequencing data on any ctenophore species and is amongst the first wave of projects to sequence an animal genome de novo solely using next-generation sequencing technologies. Description The Mnemiopsis Genome Project Portal ( http://research.nhgri.nih.gov/mnemiopsis/ ) is intended both as a resource for obtaining genomic information on Mnemiopsis through an intuitive and easy-to-use interface and as a model for developing customized Web portals that enable access to genomic data. The scope of data available through this Portal goes well beyond the sequence data available through GenBank, providing key biological information not available elsewhere, such as pathway and protein domain analyses; it also features a customized genome browser for data visualization. Conclusions We expect that the availability of these data will allow investigators to advance their own research projects aimed at understanding phylogenetic diversity and the evolution of proteins that play a fundamental role in metazoan development. The overall approach taken in the development of this Web site can serve as a viable model for disseminating data from whole-genome sequencing projects, framed in a way that best-serves the specific needs of the scientific community.

BACKGROUND:The recent expansion of whole-genome sequence data available from diverse animal lineages provides an opportunity to investigate the evolutionary origins of specific classes of human disease genes. Previous studies have observed that human disease genes are of particularly ancient origin. While this suggests that many animal species have the potential to serve as feasible models for research on genes responsible for human disease, it is unclear whether this pattern has meaningful implications and whether it prevails for every class of human disease.RESULTS:We used a comparative genomics approach encompassing a broad phylogenetic range of animals with sequenced genomes to determine the evolutionary patterns exhibited by human genes associated with different classes of disease. Our results support previous claims that most human disease genes are of ancient origin but, more importantly, we also demonstrate that several specific disease classes have a significantly large proportion of genes that emerged relatively recently within the metazoans and/or vertebrates. An independent assessment of the synonymous to non-synonymous substitution rates of human disease genes found in mammals reveals that disease classes that arose more recently also display unexpected rates of purifying selection between their mammalian and human counterparts.CONCLUSIONS:Our results reveal the heterogeneity underlying the evolutionary origins of (and selective pressures on) different classes of human disease genes. For example, some disease gene classes appear to be of uncommonly recent (i.e., vertebrate-specific) origin and, as a whole, have been evolving at a faster rate within mammals than the majority of disease classes having more ancient origins. The novel patterns that we have identified may provide new insight into cases where studies using traditional animal models were unable to produce results that translated to humans. Conversely, we note that the larger set of disease classes do have ancient origins, suggesting that many non-traditional animal models have the potential to be useful for studying many human disease genes. Taken together, these findings emphasize why model organism selection should be done on a disease-by-disease basis, with evolutionary profiles in mind.

An understanding of ctenophore biology is critical for reconstructing events that occurred early in animal evolution. Toward this goal, we have sequenced, assembled, and annotated the genome of the ctenophore Mnemiopsis leidyi . Our phylogenomic analyses of both amino acid positions and gene content suggest that ctenophores rather than sponges are the sister lineage to all other animals. Mnemiopsis lacks many of the genes found in bilaterian mesodermal cell types, suggesting that these cell types evolved independently. The set of neural genes in Mnemiopsis is similar to that of sponges, indicating that sponges may have lost a nervous system. These results present a newly supported view of early animal evolution that accounts for major losses and/or gains of sophisticated cell types, including nerve and muscle cells.

Technological advances have greatly increased the availability of human genomic sequencing. However, the capacity to analyze genomic data in a clinically meaningful way lags behind the ability to generate such data. To help address this obstacle, we reviewed all conditions with genetic causes and constructed the Clinical Genomic Database (CGD) (http://research.nhgri.nih.gov/CGD/), a searchable, freely Web-accessible database of conditions based on the clinical utility of genetic diagnosis and the availability of specific medical interventions. The CGD currently includes a total of 2,616 genes organized clinically by affected organ systems and interventions (including preventive measures, disease surveillance, and medical or surgical interventions) that could be reasonably warranted by the identification of pathogenic mutations. To aid independent analysis and optimize new data incorporation, the CGD also includes all genetic conditions for which genetic knowledge may affect the selection of supportive care, informed medical decision-making, prognostic considerations, reproductive decisions, and allow avoidance of unnecessary testing, but for which specific interventions are not otherwise currently available. For each entry, the CGD includes the gene symbol, conditions, allelic conditions, clinical categorization (for both manifestations and interventions), mode of inheritance, affected age group, description of interventions/rationale, links to other complementary databases, including databases of variants and presumed pathogenic mutations, and links to PubMed references (>20,000). The CGD will be regularly maintained and updated to keep pace with scientific discovery. Further content-based expert opinions are actively solicited. Eventually, the CGD may assist the rapid curation of individual genomes as part of active medical care.

Human immunodeficiency virus type 1 (HIV1) vectors poorly transduce rhesus hematopoietic cells due to species-specific restriction factors, including the tripartite motif-containing 5 isoform alpha (TRIM5 alpha) which targets the HIV1 capsid. We previously developed a chimeric HIV1 (chi HIV) vector system wherein the vector genome is packaged with the simian immunodeficiency virus (SIV) capsid for efficient transduction of both rhesus and human CD34(+) cells. To evaluate whether chi HIV vectors could efficiently transduce rhesus hematopoietic repopulating cells, we performed a competitive repopulation assay in rhesus macaques, in which half of the CD34(+) cells were transduced with standard SIV vectors and the other half with chi HIV vectors. As compared with SIV vectors, chi HIV vectors achieved higher vector integration, and the transgene expression rates were two- to threefold higher in granulocytes and red blood cells and equivalent in lymphocytes and platelets for 2 years. A recipient of chi HIV vector-only transduced cells reached up to 40% of transgene expression rates in granulocytes and lymphocytes and 20% in red blood cells. Similar to HIV1 and SIV vectors, chi HIV vector frequently integrated into gene regions, especially into introns. In summary, our chi HIV vector demonstrated efficient transduction for rhesus long-term repopulating cells, comparable with SIV vectors. This chi HIV vector should allow preclinical testing of HIV1-based therapeutic vectors in large animal models.

Abstract Abstract 3118 Hematopoietic stem cell (HSC)-targeted gene therapy is potentially curative for the hemoglobin disorders; however, highly efficient, lineage specific globin expression remains elusive, and large animal models thus remain important for further development toward clinical application. We previously constructed a chimeric HIV1 vector (χHIV vector) system to circumvent a species specific restriction to HIV1-based vectors wherein the HIV1 vector genome is packaged in the context of the simian immunodeficiency virus (SIV) capsid for efficient transduction of rhesus CD34+ cells in vitro (J Virol. 2009) and in vivo (ASH 2009). In this study, we sought to evaluate transduction efficiency and vector integration pattern among long-term repopulating cells in the rhesus HSC transplantation model. We followed up transgene expression rates among peripheral blood cells of three animals for 1.5–2 years. For two animals (RQ7307 and RQ7280), half of the CD34+ cells were transduced with a standard SIV vector and the other half with the χHIV vector using the same protocol. Transduced cells were transplanted into lethally irradiated rhesus macaques, as previously described (J Virol. 2009). The transgene expression rates in peripheral blood cells plateaued 3–4 months after transplantation and similar transgene expression rates continued in all cell lineages for at least 1.5 years (Figure). The χHIV vector demonstrated that 2–3 fold higher transgene expression rates were seen in granulocytes (RQ7307: 8.6±0.2% vs. 3.1±0.1%, RQ7280: 27.9±0.7% vs. 18.4±0.2%) and RBCs (RQ7307: 3.3±0.1% vs. 0.9±0.0%, RQ7280: 10.0±0.1% vs. 4.0±0.1%), and equivalent transgene expression rates in lymphocytes (RQ7307: 7.8±0.2% vs. 4.5±0.1%, RQ7280: 22.4±0.5% vs. 17.6±0.3%) and platelets (RQ7307: 3.1±0.1% vs. 2.7±0.1%, RQ7280: 12.3±0.2% vs. 16.8±0.2%), compared to the SIV vector. The average vector copy numbers in transduced cells were 4.6–5.7 for the χHIV vector and 1.5–2.0 for the SIV vector in both transplanted animals, evaluated by Southern blot analysis. We then performed transplantation of rhesus CD34+ cells which were transduced with the χHIV vector alone to evaluate transgene expression and vector integration pattern. Transgene expression rates among peripheral blood cells in this animal (RQ7387) plateaued 1–3 months after transplantation, with stable high transgene expression rates of 51.7±1.2% in granulocytes, 54.7±0.1% in lymphocytes, 22.1±0.2% in RBCs, and 19.1±0.1% platelets for 2 years after transplantation. Multi-lineage marking was observed by flow cytometric analysis. We then evaluated integration sites for the χHIV vector in the recipient of χHIV vector alone transduced cells by linear amplification mediated-PCR, using peripheral blood cells of RQ7387 in 0.5–1.5 years after transplantation. We found a total of 344 integration sites for the χHIV vector, and our data demonstrated that the χHIV vector integrated into gene regions, especially introns, when compared to the integration pattern of computer-generated random controls (p<0.001). On the other hand, our data revealed fewer integrations of the χHIV vector into ≤30kb upstream of genes (p<0.001) and into the upstream regions of transcription start sites. Most of the integration sites had low gene density (0–10 genes within 1 Mb upstream or downstream of integration sites, p<0.01), compared to that of random controls. No specific trend was noted for the number of integration sites around CpG islands and the number of CISs around integration sites. These data suggest that the χHIV vector has integration patterns comparable to HIV1 and SIV vectors. In summary, our χHIV vector shows efficient transduction for rhesus long-term repopulating cells, achieving sufficient levels for therapeutic effects in gene therapy trials for globin disorders. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in large animal models. Disclosures: No relevant conflicts of interest to declare.

We describe the creation of a specialized web-accessible database named the Pigment Cell Gene Resource, which contains information on the genetic pathways that regulate pigment cell development and function. This manually curated database is comprised of two sections, an annotated literature section and an interactive transcriptional network diagram. Initially, this database focuses on the transcription factor SOX10, which has essential roles in pigment cell development and function, but the database has been designed with the capacity to expand in the future, allowing inclusion of many more pigmentation genes. Database URL: http://research.nhgri.nih.gov/pigment_cell/

Derivation of induced pluripotent stem (iPS) cells requires the expression of defined transcription factors (among Oct3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28) in the targeted cells. Lentiviral or standard retroviral gene transfer remains the most robust and commonly used approach. Low reprogramming frequency overall, and the higher efficiency of derivation utilizing integrating vectors compared to more recent nonviral approaches, suggests that gene activation or disruption via proviral integration sites (IS) may play a role in obtaining the pluripotent phenotype. We provide for the first time an extensive analysis of the lentiviral integration profile in human iPS cells. We identified a total of 78 independent IS in eight recently established iPS cell lines derived from either human fetal fibroblasts or newborn foreskin fibroblasts after lentiviral gene transfer of Oct4, Sox2, Nanog, and Lin28. The number of IS ranged from 5 to 15 IS per individual iPS clone, and 75 IS could be assigned to a unique chromosomal location. The different iPS clones had no IS in common. Expression analysis as well as extensive bioinformatic analysis did not reveal functional concordance of the lentiviral targeted genes between the different clones. Interestingly, in six of the eight iPS clones, some of the IS were found in pairs, integrated into the same chromosomal location within six base pairs of each other or in very close proximity. Our study supports recent reports that efficient reprogramming of human somatic cells is not dependent on insertional activation or deactivation of specific genes or gene classes.