Efficient recovery of undifferentiated human embryonic stem cell cryopreserved with hydroxyethyl starch, dimethyl sulphoxide and serum replacement.

BACKGROUND: The therapeutic use of human embryonic stem cells (hESCs) is dependent on an efficient cryopreservation protocol for long-term storage. The aim of this study was to determine whether the combination of three cryoprotecting reagents using two freezing systems might improve hESC recovery rates with maintenance of hESC pluripotency properties for potential cell therapy application. METHODS: Recovery rates of hESC colonies which were frozen in three cryoprotective solutions: Me2SO/HES/SR medium, Defined-medium® and Me2SO/SFB in medium solution were evaluated in ultra-slow programmable freezing system (USPF) and a slow-rate freezing system (SRF). The hESC pluripotency properties after freezing-thawing were evaluated. RESULTS: We estimated the distribution frequency of survival colonies and observed that independent of the freezing system used (USPF or SRF) the best results were obtained with Me2SO/HES/SR as cryopreservation medium. We showed a significant hESC recovery colonies rate after thawing in Me2SO/HES/SR medium were 3.88 and 2.9 in USPF and SRF, respectively. The recovery colonies rate with Defined-medium® were 1.05 and 1.07 however in classical Me2SO medium were 0.5 and 0.86 in USPF and SRF, respectively. We showed significant difference between Me2SO/HES/SR medium×Defined-medium® and between Me2SO/HES/SR medium×Me2SO medium, for two cryopreservation systems (P<0.05). CONCLUSION: We developed an in house protocol using the combination of Me2SO/HES/SR medium and ultra-slow programmable freezing system which resulted in hESC colonies that remain undifferentiated, maintain their in vitro and in vivo pluripotency properties and genetic stability. This approach may be suitable for cell therapy studies.

Significant differences in integration sites of Moloney murine leukemia virus/Moloney murine sarcoma virus retroviral vector carrying recombinant coagulation factor IX in two human cell lines.

Ligation-mediated-PCR was performed followed by the mapping of 177 and 150 integration sites from HepG2 and Hek293 transduced with chimera vector carrying recombinant human Factor IX (rhFIX) cDNA, respectively. The sequences were analyzed for chromosome preference, CpG, transcription start site (TSS), repetitive elements, fragile sites and target genes. In HepG2, rhFIX was had an increased preference for chromosomes 6 and 17; the median distance to the nearest CpG islands was 15,240 base pairs and 37 % of the integrations occurred in RefSeq genes. In Hek293, rhFIX had an increased preference for chromosome 5; the median distance to the nearest CpG islands was 209,100 base pairs and 74 % of the integrations occurred in RefSeq genes. The integrations in both cell lines were distant from the TSS. The integration patterns associated with this vector are different in each cell line.

Optimizing Sequencing Resources in Genotyped Livestock Populations Using Linear Programming.

Low-cost genome-wide single-nucleotide polymorphisms (SNPs) are routinely used in animal breeding programs. Compared to SNP arrays, the use of whole-genome sequence data generated by the next-generation sequencing technologies (NGS) has great potential in livestock populations. However, sequencing a large number of animals to exploit the full potential of whole-genome sequence data is not feasible. Thus, novel strategies are required for the allocation of sequencing resources in genotyped livestock populations such that the entire population can be imputed, maximizing the efficiency of whole genome sequencing budgets. We present two applications of linear programming for the efficient allocation of sequencing resources. The first application is to identify the minimum number of animals for sequencing subject to the criterion that each haplotype in the population is contained in at least one of the animals selected for sequencing. The second application is the selection of animals whose haplotypes include the largest possible proportion of common haplotypes present in the population, assuming a limited sequencing budget. Both applications are available in an open source program LPChoose. In both applications, LPChoose has similar or better performance than some other methods suggesting that linear programming methods offer great potential for the efficient allocation of sequencing resources. The utility of these methods can be increased through the development of improved heuristics.

Functional analysis of HOXA10 and HOXB4 in human medulloblastoma cell lines.

Medulloblastoma (MB) is a malignant childhood brain tumor which at molecular level is classified into at least four major subtypes: WNT, SHH, group C and group D differing in response to treatment. Previous studies have associated changes in expression levels and activation of certain HOX genes with MB development. In the present study, we investigate the role of HOX genes in two attributes acquired by tumor cells: migration and proliferation potential, as well as, in vivo tumorigenic potential. We analyzed UW402, UW473, DAOY and ONS-76 human pediatric MB cell lines and cerebellum primary cultures. Two-color microarray-based gene expression analysis was used to identify differentially expressed HOX genes. Among the various HOX genes significantly overexpressed in DAOY and ONS-76 cell lines compared to UW402 and UW473 cell lines, HOXA10 and HOXB4 were selected for further analysis. The expression levels of these HOX genes were validated by real-time PCR. A mouse model was used to study the effect of the HOXA10 and HOXB4 genes on the in vivo tumorigenic potential and the in vitro proliferative and migration potential of MB cell lines. Our results show that the inhibition of HOXA10 in DAOY cell line led to increased in vitro cell migration while in vitro cell proliferation or in vivo tumorigenic potential were unaffected. We also observed that induced expression of HOXB4 in the UW473 cell line significantly reduced in vitro cell proliferation and migration capability of UW473 cells with no effect on the in vivo tumorigenicity. This suggests that HOXA10 plays a role in migration events and the HOXB4 gene is involved in proliferation and migration processes of medulloblastoma cells, however, it appears that these genes are not essential for the tumorigenic process of these cells.

Differential gene expression underlying ovarian phenotype determination in honey bee, Apis mellifera L., caste development.

Adult honey bee queens and workers drastically differ in ovary size. This adult ovary phenotype difference becomes established during the final larval instar, when massive programmed cell death leads to the degeneration of 95-99% of the ovariole anlagen in workers. The higher juvenile hormone (JH) levels in queen larvae protect the ovaries against such degeneration. To gain insights into the molecular architecture underlying this divergence critical for adult caste fate and worker sterility, we performed a microarray analysis on fourth and early fifth instar queen and worker ovaries. For the fourth instar we found nine differentially expressed genes (DEGs) with log2FC > 1.0, but this number increased to 56 in early fifth-instar ovaries. We selected 15 DEGs for quantitative PCR (RT-qPCR) analysis. Nine differed significantly by the variables caste and/or development. Interestingly, genes with enzyme functions were higher expressed in workers, while those related to transcription and signaling had higher transcript levels in queens. For the RT-qPCR confirmed genes we analyzed their response to JH. This revealed a significant up-regulation for two genes, a short chain dehydrogenase reductase (sdr) and a heat shock protein 90 (hsp90). Five other genes, including hsp60 and hexamerin 70b (hex70b), were significantly down-regulated by JH. The sdr gene had previously come up as differentially expressed in other transcriptome analyses on honey bee larvae and heat shock proteins are frequently involved in insect hormone responses, this making them interesting candidates for further functional assays.

Identifying large sets of unrelated individuals and unrelated markers.

BACKGROUND: Genetic Analyses in large sample populations are important for a better understanding of the variation between populations, for designing conservation programs, for detecting rare mutations which may be risk factors for a variety of diseases, among other reasons. However these analyses frequently assume that the participating individuals or animals are mutually unrelated which may not be the case in large samples, leading to erroneous conclusions. In order to retain as much data as possible while minimizing the risk of false positives it is useful to identify a large subset of relatively unrelated individuals in the population. This can be done using a heuristic for finding a large set of independent of nodes in an undirected graph. We describe a fast randomized heuristic for this purpose. The same methodology can also be used for identifying a suitable set of markers for analyzing population stratification, and other instances where a rapid heuristic for maximal independent sets in large graphs is needed. RESULTS: We present FastIndep, a fast random heuristic algorithm for finding a maximal independent set of nodes in an arbitrary undirected graph along with an efficient implementation in C++. On a 64 bit Linux or MacOS platform the execution time is a few minutes, even with a graph of several thousand nodes. The algorithm can discover multiple solutions of the same cardinality. FastIndep can be used to discover unlinked markers, and unrelated individuals in populations. CONCLUSIONS: The methods presented here provide a quick and efficient method for identifying sets of unrelated individuals in large populations and unlinked markers in marker panels. The C++ source code and instructions along with utilities for generating the input files in the appropriate format are available at http://taurus.ansci.iastate.edu/wiki/people/jabr/Joseph_Abraham.html.

Association between Knops blood group polymorphisms and susceptibility to malaria in an endemic area of the Brazilian Amazon.

Complement receptor 1 (CR1) gene polymorphisms that are associated with Knops blood group antigens may influence the binding of Plasmodium parasites to erythrocytes, thereby affecting susceptibility to malaria. The aim of this study was to evaluate the genotype and allele and haplotype frequencies of single-nucleotide polymorphisms (SNPs) of Knops blood group antigens and examine their association with susceptibility to malaria in an endemic area of Brazil. One hundred and twenty-six individuals from the Brazilian Amazon were studied. The CR1-genomic fragment was amplified by PCR and six SNPs and haplotypes were identified after DNA sequence analysis. Allele and haplotype frequencies revealed that the Kn(b) allele and H8 haplotype were possibly associated with susceptibility to Plasmodium falciparum. The odds ratios were reasonably high, suggesting a potentially important association between two Knops blood antigens (Kn(b) and KAM(+)) that confer susceptibility to P. falciparum in individuals from the Brazilian Amazon.

Association between Knops blood group polymorphisms and susceptibility to malaria in an endemic area of the Brazilian Amazon

Complement receptor 1 (CR1) gene polymorphisms that are associated with Knops blood group antigens may influence the binding of Plasmodium parasites to erythrocytes, thereby affecting susceptibility to malaria. The aim of this study was to evaluate the genotype and allele and haplotype frequencies of single-nucleotide polymorphisms (SNPs) of Knops blood group antigens and examine their association with susceptibility to malaria in an endemic area of Brazil. One hundred and twenty-six individuals from the Brazilian Amazon were studied. The CR1-genomic fragment was amplified by PCR and six SNPs and haplotypes were identified after DNA sequence analysis. Allele and haplotype frequencies revealed that the Kn b allele and H8 haplotype were possibly associated with susceptibility to Plasmodium falciparum. The odds ratios were reasonably high, suggesting a potentially important association between two Knops blood antigens (Kn b and KAM+) that confer susceptibility to P. falciparum in individuals from the Brazilian Amazon.