efficient-generation-hipsc-neural-lineage-specific-knockin-reporters

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

The University of Texas Health Science Center at Houston

Endosymbiosis has spread photosynthesis to many branches of the eukaryotic tree; however, the history of photosynthetic organelle (plastid) gain and loss remains controversial. Fortuitously, endosymbiosis may leave a genomic footprint through the transfer of endosymbiont genes to the "host" nucleus (endosymbiotic gene transfer, EGT). EGT can be detected through comparison of host genomes to uncover the history of past plastid acquisitions. Here we focus on a lineage of chlorophyll c-containing algae and protists ("chromalveolates") that are postulated to share a common red algal secondary endosymbiont. This plastid is originally of cyanobacterial origin through primary endosymbiosis and is closely related among the Plantae (i.e., red, green, and glaucophyte algae). To test these ideas, an automated phylogenomics pipeline was used with a novel unigene data set of 5,081 expressed sequence tags (ESTs) from the haptophyte alga Emiliania huxleyi and genome or EST data from other chromalveolates, red algae, plants, animals, fungi, and bacteria. We focused on nuclear-encoded proteins that are targeted to the plastid to express their function because this group of genes is expected to have phylogenies that are relatively easy to interpret. A total of 708 genes were identified in E. huxleyi that had a significant Blast hit to at least one other taxon in our data set. Forty-six of the alignments that were derived from the 708 genes contained at least one other chromalveolate (i.e., besides E. huxleyi), red and/or green algae (or land plants), and one or more cyanobacteria, whereas 15 alignments contained E. huxleyi, one or more other chromalveolates, and only cyanobacteria. Detailed phylogenetic analyses of these data sets turned up 19 cases of EGT that did not contain significant paralogy and had strong bootstrap support at the internal nodes, allowing us to confidently identify the source of the plastid-targeted gene in E. huxleyi. A total of 17 genes originated from the red algal lineage, whereas 2 genes were of green algal origin. Our data demonstrate the existence of multiple red algal genes that are shared among different chromalveolates, suggesting that at least a subset of this group may share a common origin.

Phylogenomic analysis identifies red algal genes of endosymbiotic origin in the chromalveolates.

Here we use phylogenomics with expressed sequence tag (EST) data from the ecologically important coccolithophore-forming alga Emiliania huxleyi and the plastid-lacking cryptophyte Goniomonas cf. pacifica to establish their phylogenetic positions in the eukaryotic tree. Haptophytes and cryptophytes are members of the putative eukaryotic supergroup Chromalveolata (chromists [cryptophytes, haptophytes, stramenopiles] and alveolates [apicomplexans, ciliates, and dinoflagellates]). The chromalveolates are postulated to be monophyletic on the basis of plastid pigmentation in photosynthetic members, plastid gene and genome relationships, nuclear "host" phylogenies of some chromalveolate lineages, unique gene duplication and replacements shared by these taxa, and the evolutionary history of components of the plastid import and translocation systems. However the phylogenetic position of cryptophytes and haptophytes and the monophyly of chromalveolates as a whole remain to be substantiated. Here we assess chromalveolate monophyly using a multigene dataset of nuclear genes that includes members of all 6 eukaryotic supergroups. An automated phylogenomics pipeline followed by targeted database searches was used to assemble a 16-protein dataset (6,735 aa) from 46 taxa for tree inference. Maximum likelihood and Bayesian analyses of these data support the monophyly of haptophytes and cryptophytes. This relationship is consistent with a gene replacement via horizontal gene transfer of plastid-encoded rpl36 that is uniquely shared by these taxa. The haptophytes + cryptophytes are sister to a clade that includes all other chromalveolates and, surprisingly, two members of the Rhizaria, Reticulomyxa filosa and Bigelowiella natans. The association of the two Rhizaria with chromalveolates is supported by the approximately unbiased (AU)-test and when the fastest evolving amino acid sites are removed from the 16-protein alignment.

Phylogenomic analysis supports the monophyly of cryptophytes and haptophytes and the association of rhizaria with chromalveolates.

Alcohol abuse during pregnancy injures the fetal brain. One of alcohol's most important neuroteratogenic effects is neuronal loss. Rat models have shown that the cerebellum becomes less vulnerable to alcohol-induced neuronal death as it matures. We determined if maturation-dependent alcohol resistance occurs in mice and compared patterns of gene expression during the alcohol resistant and sensitive periods.

Maturation-dependent alcohol resistance in the developing mouse: cerebellar neuronal loss and gene expression during alcohol-vulnerable and -resistant periods.

Fetal alcohol syndrome (FAS) stems from maternal alcohol abuse during pregnancy and is an important cause of mental retardation and hyperactivity in children. In the developing brain, alcohol can kill neurons, leading to microencephaly. However, due to their genetic makeup, some individuals are less vulnerable than others to alcohol's neurotoxic effects. Animal studies have demonstrated that one particular gene, neuronal nitric oxide synthase (nNOS), protects developing neurons in vivo against alcohol-induced death. We utilized pharmacologic techniques to demonstrate that nNOS protects neurons against alcohol toxicity by activating the NO-cGMP-PKG signaling pathway. Cerebellar granule cell cultures derived from mice carrying a null mutation for nNOS (nNOS-/- mice) were substantially more vulnerable than cultures from wild-type mice to alcohol-induced cell death. However, activation of the pathway at sites downstream of nNOS protected the cultures against alcohol toxicity. Conversely, blockade of the pathway rendered wild-type cultures vulnerable to alcohol-induced death. We further identified NF-kappaB as the downstream effector through which nNOS and the NO-cGMP-PKG pathway signal their neuroprotective effects. Tumor necrosis factor-alpha (TNF-alpha), which activates NF-kappaB, ameliorated alcohol-induced cell death in nNOS-/- and wild-type cultures, while an NF-kappaB inhibitor (NFi) blocked the protective effects of TNF-alpha and worsened alcohol-induced cell death. Furthermore, NFi blocked the protective effects of NO-cGMP-PKG pathway activators, demonstrating that NF-kappaB is downstream of the NO-cGMP-PKG pathway. As wild-type neurons matured in culture, they became resistant to alcohol toxicity. However, this maturation-dependent alcohol resistance did not occur in nNOS-/- mice and could be reversed in wild-type mice with NFi, demonstrating that nitric oxide and NF-kappaB are crucial for the development of alcohol resistance with age. Thus, nNOS protects developing neurons against alcohol toxicity by activating the NO-cGMP-PKG-NF-kappaB pathway and is crucial for the acquisition of maturation-dependent alcohol resistance.

The protective effect of neuronal nitric oxide synthase (nNOS) against alcohol toxicity depends upon the NO-cGMP-PKG pathway and NF-kappaB.

The role of serum tumour markers is to reveal tumours not yet visible by imaging techniques. Here we examine the use of serum thymidine kinase 1 protein (STK1) in health screening.

Serological thymidine kinase 1 (STK1) indicates an elevated risk for the development of malignant tumours.

Acute liver failure is a devastating illness of various causes with considerable mortality. Hepatic stimulator substance (HSS) has been suggested for use as a protective agent against acute hepatic injury induced by chemical poisons because it has a variety of biological activities. However, the mechanism whereby HSS protects against hepatotoxins is poorly understood. In this study, we established a hepatic gene transfer system via hydrodynamic tail vein injection to deliver a naked plasmid containing the human HSS gene (hHSS) and analyzed HSS-mediated protection of the liver during fulminant hepatic failure (FHF) induced by D-galactosamine (D-gal) and lipopolysaccharide (LPS). The results showed that the reporter gene, enhanced green fluorescent protein, was efficiently expressed in the liver of BALB/c mice. Hydrodynamic-based transfection of hHSS yielded a 70% survival rate compared with 36.7% for the control group at 24 h after D-gal/LPS treatment. In addition, hHSS expression preserved liver morphology and function. It is noteworthy that hHSS hydrodynamic-based transfer ameliorated indices of the mitochondrial permeability transition (MPT) resulting from the toxic effects of d-gal/LPS on the liver such as mitochondrial swelling, mitochondrial transmembrane potential disruption, and cytochrome c translocation. Furthermore, mitochondrial morphology and ATP levels were maintained in hHSS-administered mice. HSS-mediated protection was similar to that observed with the MPT inhibitor N-methyl-4-isoleucine-cyclosporin (NIM811), indicating a possible role for HSS in the regulation of MPT. In conclusion, a single dose of hHSS plasmid protected mice from FHF, and this hepatoprotective effect seemed to correlate with the inhibition of MPT.

Administration of naked plasmid encoding hepatic stimulator substance by hydrodynamic tail vein injection protects mice from hepatic failure by suppressing the mitochondrial permeability transition.

A simple and selective method was developed for the simultaneous determination of tetrandrine and fangchinoline in herbal medicine by HPLC with electrochemical detection (ECD) on a bare glassy carbon electrode. The HPLC separation and ECD conditions have been optimized. The separation was carried out on a WondaSil C18-WR column (4.6 mm × 250 mm, 5 μm), with the mobile phase of acetonitrile-ammonium acetate buffer (pH 6.5; 40 mM) (32:68, v/v) using an isocratic elution at the flow rate of 0.5 mL/min. The electrochemical detection potential was set at +0.9 V. The obtained LODs for tetrandrine and fangchinoline were 0.26 and 0.27 μmol/L, respectively. The method was successfully applied to the analysis of tetrandrine and fangchinoline contents in Stephania tetrandra S. Moore. It has been demonstrated that the LC-ECD method is an excellent technique for analysis of the herbal medicine. The mean recoveries were in the range of 95-105%, while the precision expressed as repetition of peak area was lower than 2.7%.

Simultaneous determination of tetrandrine and fangchinoline in herbal medicine Stephania tetrandra S. Moore by liquid chromatography with electrochemical detection.

Genetic modification is an indispensable tool to study gene function in normal development and disease. The recent breakthrough of creating human induced pluripotent stem cells (iPSCs) by defined factors (Takahashi et al., Cell 131:861-872, 2007) provides a renewable source of patient autologous cells that not only retain identical genetic information but also give rise to many cell types of the body including neurons and glia. Meanwhile, the rapid advancement of genome modification tools such as gene targeting by homologous recombination (Capecchi, Nat Rev Genet 6:507-512, 2005) and genome editing tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, TALENs (Transcription activator-like effector nucleases), and ZFNs (Zinc finger nucleases) (Wang et al., Cell 153:910-918, 2013; Mali et al., Science 339:823-826, 2013; Hwang et al., Nat Biotechnol 31:227-229, 2013; Friedland et al., Nat Methods 10(8):741-743, 2013; DiCarlo et al., Nucleic Acids Res 41:4336-4343, 2013; Cong et al., Science 339:819-823, 2013) has greatly accelerated the development of human genome manipulation at the molecular level. This chapter describes the protocols for making neural lineage reporter lines using homologous recombination and the CRISPR/Cas system-mediated genome editing, including construction of targeting vectors, guide RNAs, transfection into hPSCs, and selection and verification of successfully targeted clones. This method can be applied to various needs of hPSC genetic engineering at high efficiency and high reliability.

Genetic Modification in Human Pluripotent Stem Cells by Homologous Recombination and CRISPR/Cas9 System.

Down's syndrome (DS), caused by trisomy of human chromosome 21, is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons, and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo. We also observed abnormal gene expression profiles from DS astroglia. Finally, we show that the FDA-approved antibiotic drug, minocycline, partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B, GFAP, inducible nitric oxide synthase, and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug.

Role of astroglia in Down's syndrome revealed by patient-derived human-induced pluripotent stem cells.

Glucocorticoids (GC) are increasingly being used to treat chronic rhinosinusitis, its efficacy and safety are the focus of attention. This article will be divided into chronic rhinosinusitis with nasal polyps and without nasal polyps two subtypes discussed nasal and oral glucocorticoids in the clinical therapeutic efficacy and medication safety.

Article	Year
Phylogenomic analysis identifies red algal genes of endosymbiotic origin in the chromalveolates. Molecular biology and evolution\| PubMed ID: 16357039	2006
Phylogenomic analysis supports the monophyly of cryptophytes and haptophytes and the association of rhizaria with chromalveolates. Molecular biology and evolution\| PubMed ID: 17488740	2007
Maturation-dependent alcohol resistance in the developing mouse: cerebellar neuronal loss and gene expression during alcohol-vulnerable and -resistant periods. Alcoholism, clinical and experimental research\| PubMed ID: 18565154	2008
The protective effect of neuronal nitric oxide synthase (nNOS) against alcohol toxicity depends upon the NO-cGMP-PKG pathway and NF-kappaB. Neurotoxicology\| PubMed ID: 18824032	2008
Serological thymidine kinase 1 (STK1) indicates an elevated risk for the development of malignant tumours. Anticancer research\| PubMed ID: 19192647	2008
Administration of naked plasmid encoding hepatic stimulator substance by hydrodynamic tail vein injection protects mice from hepatic failure by suppressing the mitochondrial permeability tra... The Journal of pharmacology and experimental therapeutics\| PubMed ID: 21613410	2011
Simultaneous determination of tetrandrine and fangchinoline in herbal medicine Stephania tetrandra S. Moore by liquid chromatography with electrochemical detection. Journal of pharmaceutical and biomedical analysis\| PubMed ID: 22196802	2012
Genetic Modification in Human Pluripotent Stem Cells by Homologous Recombination and CRISPR/Cas9 System. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 24615461	2014
Role of astroglia in Down's syndrome revealed by patient-derived human-induced pluripotent stem cells. Nature communications\| PubMed ID: 25034944	2014
[The role of glucocorticoids in the treatment of chornic rhinosinusitis]. Lin chuang er bi yan hou tou jing wai ke za zhi = Journal of clinical otorhinolaryngology, head, and neck surgery\| PubMed ID: 25185299	2014

Shenglan Li

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