PiggyBac Mediated Multiplex Gene Transfer in Mouse Embryonic Stem Cell

PiggyBac system has been shown to have a high efficiency to mediate gene transfer. However, there are no reports on its efficiency to mediate multiplex transgenes in mouse embryonic stem cells. Here we first established an immortalized feeder cell line by introducing four antibiotic resistance genes simultaneously into the original SNL 76/7 feeder cell line utilizing the PiggyBac system. This is the feeder cell line with the most diverse types of antibiotic resistance genes reported so far, which will enable researchers to perform simultaneous multiplex gene transfer or gene targeting experiments in ES cells. With such feeder cell line, we were able to quantitatively characterize the transposition efficiency of PiggyBac system in mouse ES cells using five transposons carrying different inducible fluorescence proteins and antibiotic resistance genes, and the efficiency ranged from about 2% for one transposon to 0.5% for five transposons. The highly efficient multiplex gene transfer mediated by PiggyBac will no doubt provide researchers with more choices in biomedical research and development.     

NPM1 Deletion Is Associated with Gross Chromosomal Rearrangements in Leukemia

Background

NPM1 gene at chromosome 5q35 is involved in recurrent translocations in leukemia and lymphoma. It also undergoes mutations in 60% of adult acute myeloid leukemia (AML) cases with normal karyotype. The incidence and significance of NPM1 deletion in human leukemia have not been elucidated.

Methodology and Principal Findings

Bone marrow samples from 145 patients with myelodysplastic syndromes (MDS) and AML were included in this study. Cytogenetically 43 cases had isolated 5q-, 84 cases had 5q- plus other changes and 18 cases had complex karyotype without 5q deletion. FISH and direct sequencing investigated the NPM1 gene. NPM1 deletion was an uncommon event in the “5q- syndrome” but occurred in over 40% of cases with high risk MDS/AML with complex karyotypes and 5q loss. It originated from large 5q chromosome deletions. Simultaneous exon 12 mutations were never found. NPM1 gene status was related to the pattern of complex cytogenetic aberrations. NPM1 haploinsufficiency was significantly associated with monosomies (p<0.001) and gross chromosomal rearrangements, i.e., markers, rings, and double minutes (p<0.001), while NPM1 disomy was associated with structural changes (p = 0.013). Interestingly, in complex karyotypes with 5q- TP53 deletion and/or mutations are not specifically associated with NPM1 deletion.

Conclusions and Significance

NPM1/5q35 deletion is a consistent event in MDS/AML with a 5q-/-5 in complex karyotypes. NPM1 deletion and NPM1 exon 12 mutations appear to be mutually exclusive and are associated with two distinct cytogenetic subsets of MDS and AML.     

In Vivo Fate Imaging of Intracerebral Stem Cell Grafts in Mouse Brain

We generated transgenic human neural stem cells (hNSCs) stably expressing the reporter genes Luciferase for bioluminescence imaging (BLI) and GFP for fluorescence imaging, for multimodal imaging investigations. These transgenic hNSCs were further labeled with a clinically approved perfluoropolyether to perform parallel ¹⁹F MRI studies. In vitro validation demonstrated normal cell proliferation and differentiation of the transgenic and additionally labeled hNSCs, closely the same as the wild type cell line, making them suitable for in vivo application. Labeled and unlabeled transgenic hNSCs were implanted into the striatum of mouse brain. The time profile of their cell fate after intracerebral grafting was monitored during nine days following implantation with our multimodal imaging approach, assessing both functional and anatomical condition. The ¹⁹F MRI demarcated the graft location and permitted to estimate the cell number in the graft. BLI showed a pronounce cell loss during this monitoring period, indicated by the decrease of the viability signal. The in vivo obtained cell fate results were further validated and confirmed by immunohistochemistry. We could show that the surviving cells of the graft continued to differentiate into early neurons, while the severe cell loss could be explained by an inflammatory reaction to the graft, showing the graft being surrounded by activated microglia and macrophages. These results are different from earlier cell survival studies of our group where we had implanted the identical cells into the same mouse strain but in the cortex and not in the striatum. The cortical transplanted cells did not show any loss in viability but only pronounced and continuous neuronal differentiation.     

Yy1 Gene Dosage Effect and Bi-Allelic Expression of Peg3

In the current study, we tested the in vivo effects of Yy1 gene dosage on the Peg3 imprinted domain with various breeding schemes utilizing two sets of mutant alleles. The results indicated that a half dosage of Yy1 coincides with the up-regulation of Peg3 and Zim1, suggesting a repressor role of Yy1 in this imprinted domain. This repressor role of Yy1 is consistent with the observations derived from previous in vitro studies. The current study also provided an unexpected observation that the maternal allele of Peg3 is also normally expressed, and thus the expression of Peg3 is bi-allelic in the specific areas of the brain, including the choroid plexus, the PVN (Paraventricular Nucleus) and the SON (Supraoptic Nucleus) of the hypothalamus. The exact roles of the maternal allele of Peg3 in these cell types are currently unknown, but this new finding confirms the previous prediction that the maternal allele may be functional in specific cell types based on the lethality associated with the homozygotes for several mutant alleles of the Peg3 locus. Overall, these results confirm the repressor role of Yy1 in the Peg3 domain and also provide a new insight regarding the bi-allelic expression of Peg3 in mouse brain.     

SERPINA2 Is a Novel Gene with a Divergent Function from SERPINA1

Serine protease inhibitors (SERPINs) are a superfamily of highly conserved proteins that play a key role in controlling the activity of proteases in diverse biological processes. The SERPIN cluster located at the 14q32.1 region includes the gene coding for SERPINA1, and a highly homologous sequence, SERPINA2, which was originally thought to be a pseudogene. We have previously shown that SERPINA2 is expressed in different tissues, namely leukocytes and testes, suggesting that it is a functional SERPIN. To investigate the function of SERPINA2, we used HeLa cells stably transduced with the different variants of SERPINA2 and SERPINA1 (M1, S and Z) and leukocytes as the in vivo model. We identified SERPINA2 as a 52 kDa intracellular glycoprotein, which is localized at the endoplasmic reticulum (ER), independently of the variant analyzed. SERPINA2 is not significantly regulated by proteasome, proposing that ER localization is not due to misfolding. Specific features of SERPINA2 include the absence of insoluble aggregates and the insignificant response to cell stress, suggesting that it is a non-polymerogenic protein with divergent activity of SERPINA1. Using phylogenetic analysis, we propose an origin of SERPINA2 in the crown of primates, and we unveiled the overall conservation of SERPINA2 and A1. Nonetheless, few SERPINA2 residues seem to have evolved faster, contributing to the emergence of a new advantageous function, possibly as a chymotrypsin-like SERPIN. Herein, we present evidences that SERPINA2 is an active gene, coding for an ER-resident protein, which may act as substrate or adjuvant of ER-chaperones.     

Gene Expression Analysis of PTEN Positive Glioblastoma Stem Cells Identifies DUB3 and Wee1 Modulation in a Cell Differentiation Model

The term astrocytoma defines a quite heterogeneous group of neoplastic diseases that collectively represent the most frequent brain tumors in humans. Among them, glioblastoma multiforme represents the most malignant form and its associated prognosis is one of the poorest among tumors of the central nervous system. It has been demonstrated that a small population of tumor cells, isolated from the brain neoplastic tissue, can reproduce the parental tumor when transplanted in immunodeficient mouse. These tumor initiating cells are supposed to be involved in cancer development and progression and possess stem cell-like features; like their normal counterpart, these cells remain quiescent until they are committed to differentiation. Many studies have shown that the role of the tumor suppressor protein PTEN in cell cycle progression is fundamental for tumor dynamics: in low grade gliomas, PTEN contributes to maintain cells in G1 while the loss of its activity is frequently observed in high grade gliomas. The mechanisms underlying the above described PTEN activity have been studied in many tumors, but those involved in the maintenance of tumor initiating cells quiescence remain to be investigated in more detail. The aim of the present study is to shed light on the role of PTEN pathway on cell cycle regulation in Glioblastoma stem cells, through a cell differentiation model. Our results suggest the existence of a molecular mechanism, that involves DUB3 and WEE1 gene products in the regulation of Cdc25a, as functional effector of the PTEN/Akt pathway.     

Defects in DNA Lesion Bypass Lead to Spontaneous Chromosomal Rearrangements and Increased Cell Death

Rev3 polymerase and Mph1 DNA helicase participate in error-prone and error-free pathways, respectively, for the bypassing of template lesions during DNA replication. Here we have investigated the role of these pathways and their genetic interaction with recombination factors, other nonreplicative DNA helicases, and DNA damage checkpoint components in the maintenance of genome stability, viability, and sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS). We find that cells lacking Rev3 and Mph1 exhibit a synergistic, Srs2-dependent increase in the rate of accumulating spontaneous, gross chromosomal rearrangements, suggesting that the suppression of point mutations by deletion of REV3 may lead to chromosomal rearrangements. While mph1Δ is epistatic to homologous recombination (HR) genes, both Rad51 and Rad52, but not Rad59, are required for normal growth of the rev3Δ mutant and are essential for survival of rev3Δ cells during exposure to MMS, indicating that Mph1 acts in a Rad51-dependent, Rad59-independent subpathway of HR-mediated lesion bypass. Deletion of MPH1 helicase leads to synergistic DNA damage sensitivity increases in cells with chl1Δ or rrm3Δ helicase mutations, whereas mph1Δ is hypostatic to sgs1Δ. Previously reported slow growth of mph1Δ srs2Δ cells is accompanied by G₂/M arrest and fully suppressed by disruption of the Mec3-dependent DNA damage checkpoint. We propose a model for replication fork rescue mediated by translesion DNA synthesis and homologous recombination that integrates the role of Mph1 in unwinding D loops and its genetic interaction with Rev3 and Srs2-regulated pathways in the suppression of spontaneous genome rearrangements and in mutation avoidance.Nonreplicative DNA helicases play an important role in the maintenance of genome stability from bacteria to humans, most likely by affecting the formation and/or resolution of recombination intermediates and by facilitating replication fork progression through chromosomal regions with a propensity to adopt unusual DNA structures or those bound by proteins. In Saccharomyces cerevisiae, this group of DNA helicases includes the 3′-to-5′ helicases Sgs1 and Srs2 and the 5′-to-3′ DNA helicase Rrm3. In the absence of any two of these three helicases, unresolved recombination intermediates accumulate and lead to extremely slow growth that is fully suppressed by deletion of genes encoding early homologous recombination (HR) factors (4, 6, 17, 20, 37, 46). In the absence of Sgs1, cells exhibit increased rates of mitotic recombination, frequent chromosome missegregation, accumulation of extrachromosomal ribosomal DNA (rDNA) circles, and increased rates of gross chromosomal rearrangements (GCRs) involving nonhomologous chromosomes (5, 24, 25, 38, 40, 43, 49, 50). Based on the increased crossover frequency during HO endonuclease-induced double-strand breaks (DSBs) in cells lacking Sgs1, it has also been proposed that Sgs1 may function in decatenation of Holliday junctions (HJs) to yield noncrossovers (12, 22). Like Sgs1, Srs2 acts to favor noncrossover outcomes during DSB repair but appears to act earlier than Sgs1 in regulating recombination outcomes through its ability to dislodge Rad51 from recombinogenic 3′ overhangs, thereby promoting a noncrossover synthesis-dependent single-strand annealing (SDSA) pathway (12, 33, 35). In contrast, Rrm3 has not been implicated in DNA repair but is thought to be important for avoidance of recombination substrate formation by removal of DNA protein complexes in certain chromosomal locations, such as chromosome ends and replication fork barriers at the rDNA locus, thus facilitating replication fork progression (13, 14).In addition to Sgs1, Rrm3, and Srs2, the yeast genome encodes two other nonreplicative DNA helicases with proposed functions in DNA repair, Mph1 and Chl1. Mph1 possesses 3′-to-5′ helicase activity, and its ATPase activity requires a relatively long fragment of single-stranded DNA (ssDNA) (≥40 nucleotides [nt]) for full activity in vitro (32). Mph1 is also necessary for resistance to the DNA damaging agents methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4-NQO) and suppresses spontaneous mutations toward canavanine resistance (3, 41). The modest mutator phenotype of the mph1Δ mutant is enhanced by additional mutations in base excision repair (apn1Δ and apn2Δ) and is suppressed by mutations in translesion DNA synthesis (TLS) (rev3Δ) (36, 41). These findings, in combination with the observation of an epistatic relationship between mph1Δ and homologous recombination mutations, have led to the proposal that Mph1 may act in Rad52-dependent, error-free bypassing of DNA lesions (41). Like the 3′-to-5′ DNA helicases Sgs1 and Srs2, Mph1 was recently shown to affect crossover frequency during repair of an HO endonuclease-induced DNA DSB, favoring noncrossovers as the outcome (33). The authors showed that Mph1 can unwind intermediates of homologous recombination in vitro, specifically D loops that are thought to form early during homologous recombination when a homoduplex is invaded by a Rad51 filament. While Srs2 has been shown to be able to disassemble Rad51 filaments in vitro, it does not appear to possess Mph1''s ability to dissociate D loops once they have formed (19, 47).Although Chl1 has been shown to be required for the establishment of sister chromatid cohesion, a possible role in DNA repair by homologous recombination has also been proposed (11, 28, 30, 42). While Chl1 possesses a conserved helicase domain, helicase activity has so far been shown only for its putative human homolog, hCHLR1 (10).To further elucidate the functional interaction between nonreplicative DNA helicases and DNA repair pathways, we generated a series of mutants with combinations of mph1Δ, chl1Δ, rrm3Δ, srs2Δ, and sgs1Δ mutations and mutations in translesion DNA synthesis (TLS), base excision repair (BER), homologous recombination (HR), and DNA damage checkpoints. In addition to synthetic fitness defects due to aberrant HR and checkpoint activation, we identified epistatic and synergistic relationships with regard to fitness, the accumulation of gross chromosomal rearrangements (GCRs), and sensitivity to DNA damage. We propose that Mph1 functions in a Rad51-dependent, Rad59-independent pathway of HR for DNA lesion bypass and interacts genetically with REV3 in the suppression of gross chromosomal rearrangements.     

Gemini,a Bifunctional Enzymatic and Fluorescent Reporter of Gene Expression

Background

The development of collections of quantitatively characterized standard biological parts should facilitate the engineering of increasingly complex and novel biological systems. The existing enzymatic and fluorescent reporters that are used to characterize biological part functions exhibit strengths and limitations. Combining both enzymatic and fluorescence activities within a single reporter protein would provide a useful tool for biological part characterization.

Methodology/Principal Findings

Here, we describe the construction and quantitative characterization of Gemini, a fusion between the β-galactosidase (β-gal) α-fragment and the N-terminus of full-length green fluorescent protein (GFP). We show that Gemini exhibits functional β-gal activity, which we assay with plates and fluorometry, and functional GFP activity, which we assay with fluorometry and microscopy. We show that the protein fusion increases the sensitivity of β-gal activity and decreases the sensitivity of GFP.

Conclusions/Significance

Gemini is therefore a bifunctional reporter with a wider dynamic range than the β-gal α-fragment or GFP alone. Gemini enables the characterization of gene expression, screening assays via enzymatic activity, and quantitative single-cell microscopy or FACS via fluorescence activity. The analytical flexibility afforded by Gemini will likely increase the efficiency of research, particularly for screening and characterization of libraries of standard biological parts.     

Gene Expression Profiling Reveals a Novel Regulatory Role for Sox21 Protein in Mouse Trophoblast Stem Cell Differentiation

Appropriate self-renewal and differentiation of trophoblast stem cells (TSCs) are key factors for proper placental development and function and, in turn, for appropriate in utero fetal growth. To identify novel TSC-specific genes, we performed genome-wide expression profiling of TSCs, embryonic stem cells, epiblast stem cells, and mouse embryo fibroblasts, derived from mice of the same genetic background. Our analysis revealed a high expression of Sox21 in TSCs compared with other cell types. Sox21 levels were high in undifferentiated TSCs and were dramatically reduced upon differentiation. In addition, modulation of Sox21 expression in TSCs affected lineage-specific differentiation, based on both marker analysis and functional assessment. Our results implicate Sox21 specifically in the promotion of spongiotrophoblast and giant cell differentiation and establish a new mechanism through which trophoblast sublineages are specified.     

Deletion of Fgfr1 in Osteoblasts Enhances Mobilization of EPCs into Peripheral Blood in a Mouse Endotoxemia Model

Endothelial progenitor cells (EPCs) contribute to neovascularization and vascular repair, and may exert a beneficial effect on the clinical outcome of sepsis. Osteoblasts act as a component of “niche” in bone marrow, which provides a nest for stem/progenitor cells and are involved in the formation and maintenance of stem/progenitor cells. Fibroblast growth factor receptor 1 (FGFR1) can regulate osteoblast activity and influence bone mass. So we explored the role of FGFR1 in EPC mobilization. Male mice with osteoblast-specific knockout of Fgfr1 (Fgfr1^fl/fl;OC-Cre) and its wild-type littermates (Fgfr1^fl/fl) were used in this study. Mice intraperitoneally injected with lipopolysaccharide (LPS) were used to measure the number of circulating EPCs in peripheral blood and serum stromal cell-derived factor 1α (SDF-1α). The circulating EPC number and the serum level of SDF-1α were significantly higher in Fgfr1^fl/fl;OC-Cre mice than those in Fgfr1^fl/fl mice after LPS injection. In cell culture system, SDF-1α level was also significantly higher in Fgfr1^fl/fl;OC-Cre osteoblasts compared with that in Fgfr1^fl/fl osteoblasts after LPS treatment. TRAP staining showed that there was no significant difference between the osteoclast activity of septic Fgfr1^fl/fland Fgfr1^fl/fl;OC-Cre mice. This study suggests that targeted deletion of Fgfr1 in osteoblasts enhances mobilization of EPCs into peripheral blood through up-regulating SDF-1α secretion from osteoblasts.