A Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout Clones

Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines.

Regulation of Nuclear Factor κB (NF-κB) Transcriptional Activity via p65 Acetylation by the Chaperonin Containing TCP1 (CCT)

The NF-κB family member p65 is central to inflammation and immunity. The purpose of this study was to identify and characterize evolutionary conserved genes modulating p65 transcriptional activity. Using an RNAi screening approach, we identified chaperonin containing TCP1 subunit η (CCTη) as a regulator of Drosophila NF-κB proteins, Dorsal and Dorsal-related immunity factor (Dif). CCTη was also found to regulate NF-κB-driven transcription in mammalian cells, acting in a promoter-specific context, downstream of IκB kinase (IKK). CCTη knockdown repressed IκBα and CXCL2/MIP2 transcription during the early phase of NF-κB activation while impairing the termination of CCL5/RANTES and CXCL10/IP10 transcription. The latter effect was associated with increased DNA binding and reduced p65 acetylation, presumably by altering the activity of histone acetyltransferase CREB-binding protein (CBP). We identified p65 lysines (K) 122 and 123 as target residues mediating the CCTη-driven termination of NF-κB-dependent transcription. We propose that CCTη regulates NF-κB activity in a manner that resolves inflammation.

Amino ozonides exhibit in vitro activity against Echinococcus multilocularis metacestodes.

Artemisinin is an antimalarial sesquiterpene lactone that contains a 1,2,4-trioxane heterocycle. Dihydroartemisinin and artesunate demonstrated activity against Echinococcus multilocularis metacestodes in vitro but were not effective in a mouse model. In this study, the in vitro effects of a small library of synthetic ozonides (1,2,4-trioxolanes) were investigated. Initial compound screening against E. multilocularis metacestodes was performed at 20μM, and selected ozonides were further assessed in dose-response studies in metacestode cultures and mammalian cells. Transmission electron microscopy (TEM) was employed to characterise compound-induced structural alterations. At 20μM, the most potent ozonides (OZ401, OZ455, OZ491 and OZ494) led to death of ca. 60-100% of the parasites. Subsequent dose-response experiments demonstrated that OZ401, OZ455 and OZ491, which contain an aminopropylether substructure, were the most potent, with 50% inhibitory concentrations ranging from 11μM to 14μM. Cytotoxicity for these three ozonides, assessed in human foreskin fibroblasts, rat hepatoma cells and green monkey epithelial kidney (Vero) cells, was evident only at high concentrations. TEM demonstrated that OZ401 and OZ491 treatment induced considerable metabolic impairment in metacestodes at 1 day post exposure. At Day 3 post exposure, the germinal layer was severely distorted, although some intact cells were still visible, demonstrating that not all cell types in the parasite tissue were equally affected. Complete destruction of the germinal layer was noted at 5 days post exposure. Synthetic ozonides could represent interesting leads that will be further investigated in a suitable in vivo model of E. multilocularis infection.

A synthetic biology-based device prevents liver injury in mice

BACKGROUND & AIMS The liver performs a panoply of complex activities coordinating metabolic, immunologic and detoxification processes. Despite the liver's robustness and unique self-regeneration capacity, viral infection, autoimmune disorders, fatty liver disease, alcohol abuse and drug-induced hepatotoxicity contribute to the increasing prevalence of liver failure. Liver injuries impair the clearance of bile acids from the hepatic portal vein which leads to their spill over into the peripheral circulation where they activate the G-protein-coupled bile acid receptor TGR5 to initiate a variety of hepatoprotective processes. METHODS By functionally linking activation of ectopically expressed TGR5 to an artificial promoter controlling transcription of the hepatocyte growth factor (HGF), we created a closed-loop synthetic signalling network that coordinated liver injury-associated serum bile acid levels to expression of HGF in a self-sufficient, reversible and dose-dependent manner. RESULTS After implantation of genetically engineered human cells inside auto-vascularizing, immunoprotective and clinically validated alginate-poly-(L-lysine)-alginate beads into mice, the liver-protection device detected pathologic serum bile acid levels and produced therapeutic HGF levels that protected the animals from acute drug-induced liver failure. CONCLUSIONS Genetically engineered cells containing theranostic gene circuits that dynamically interface with host metabolism may provide novel opportunities for preventive, acute and chronic healthcare. LAY SUMMARY Liver diseases leading to organ failure may go unnoticed as they do not trigger any symptoms or significant discomfort. We have designed a synthetic gene circuit that senses excessive bile acid levels associated with liver injuries and automatically produces a therapeutic protein in response. When integrated into mammalian cells and implanted into mice, the circuit detects the onset of liver injuries and coordinates the production of a protein pharmaceutical which prevents liver damage.

High throughput determination of sequence-function relationships in protein and RNA

Thesis (Ph.D.)--University of Washington, 2016-06

Comparative proteomic analysis of GS-NSO murine myeloma cell lines with varying recombinant monoclonal antibody production rate

We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NSO) cells. Four homogeneous NSO cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab clatasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production. (C) 2004 Wiley Periodicals, Inc.

Acute cadmium chloride administration induces hepatic and renal CYP2A5 mRNA, protein and activity in the mouse: involvement of transcription factor NRF2

Modulation of the cytochrome P450 (CYP) monooxygenase system by cadmium was investigated in male, adult DBA/2J mice treated with a single dose (16 mumol/kg body weight, i.p.) of cadmium chloride (CdCl2). Total CYP content of liver and kidney microsomes decreased maximally (56% and 85%, respectively) 24 and 18 h, respectively, after CdCl2 treatment. Progressive increases of hepatic coumarin 7-hydroxylase (COH) activity; indicative of CYP2A5 activity, relative to the total CYP content were seen at 8 h (2-fold), 12 h (3-fold), 18 h (12-fold), and 24 h (15-fold). Similar changes were seen in the kidney. Liver and kidney CYP2A5 mRNA levels increased maximally 12 and 4 h after treatment and decreased to almost half 6 h later. In contrast, kidney and liver CYP2A5 protein levels increased maximally at 18 and 24 h. The CYP2A5 mRNA levels in the kidney and liver increased after Cd treatment in Nrf2 +/+ but not in Nrf2 -/- mouse. This study demonstrates that hepatic and kidney CYP2A5 is upregulated by cadmium with a somewhat faster response in the kidney than the liver. The strong upregulation of the CYP2A5 both at mRNA and enzyme activity levels, with a simultaneous decrease in the total CYP concentration suggest an unusual mode of regulation of CYP2A5 in response to cadmium exposure, amongst the CYP enzymes. The observed decrease in the mRNA but not in protein levels after maximal induction may suggest involvement of post-trancriptional mechanisms in the regulation. Upregulation of CYP2A5 by cadmium in the Nrf2 +/+ mice but not in the Nrf2 -/- mice indicates a role for this transcription factor in the regulation. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

tRNASer(CGA) differentially regulates expression of wild-type and codon-modified papillomavirus L1 genes

Exogenous transfer RNAs (tRNAs) favor translation of bovine papillomavirus 1 wild-type (wt) L1 mRNA in in vitro translation systems (Zhou et al. 1999, J. Virol., 73, 4972-4982). We, therefore, investigated whether papillomavirus (PV) wt L1 protein expression could be enhanced in eukaryotic cells following exogenous tRNA supplementation. Both Chinese hamster ovary (CHO) and Cos1 cells, transfected with PV1 wt L1 genes, effectively transcribed the genes but did not translate them. However, L1 protein translation was demonstrated following co-transfection with the L1 gene and a gene expressing tRNA(Ser)(CGA). Cell lines, stably transfected with a bovine papillomavirus 1 (BPV1) wt L1 expression construct, produced L1 protein after the transfection of the tRNA(Ser)(CGA) gene, but not following the transfection with basal vectors, suggesting that tRNA(Ser)(CGA) gene enhanced wt L1 translation as a result of endogenous tRNA alterations and phosphorylation of translation initiation factors elF4E and elF2alpha in the tRNA(Ser)(CGA) transfected L1 cell lines. The tRNA(Ser)(CGA) gene expression significantly reduced translation of L1 proteins expressed from codon-modified (HB) PV L1 genes utilizing mammalian preferred codons, but had variable effects on translation of green fluorescent proteins (GFPs) expressed from six serine GFP variants. The changes of tRNA pools appear to match the codon composition of PV wt and HB L1 genes and serine GFP variants to regulate translation of their mRNAs. These findings demonstrate for the first time in eukaryotic cells that translation of the target genes can be differentially influenced by the provision of a single tRNA expression construct.

Novel roles for the AIDA adhesion from diarrheagenic Escherichia coli: Cell aggregation and biofilm formation

Diarrhea-causing Escherichia coli strains are responsible for numerous cases of gastrointestinal disease and constitute a serious health problem throughout the world. The ability to recognize and attach to host intestinal surfaces is an essential step in the pathogenesis of such strains. AIDA is a potent bacterial adhesin associated with some diarrheagenic E. coli strains. AIDA mediates bacterial attachment to a broad variety of human and other mammalian cells. It is a surface-displayed autotransporter protein and belongs to the selected group of bacterial glycoproteins; only the glycosylated form binds to mammalian cells. Here, we show that AIDA possesses self-association characteristics and can mediate autoaggregation of E. coli cells. We demonstrate that intercellular AIDA-AIDA interaction is responsible for bacterial autoaggregation. Interestingly, AIDA-expressing cells can interact with antigen 43 (Ag43) -expressing cells, which is indicative of an intercellular AIDA-Ag43 interaction. Additionally, AIDA expression dramatically enhances biofilm formation by E. coli on abiotic surfaces in How chambers.

Design of artificial myocardial microtissues

Cultivation technologies promoting organization of mammalian cells in three dimensions are essential for gene-function analyses as well as drug testing and represent the first step toward the design of tissue replacements and bioartificial organs. Embedded in a three-dimensional environment, cells are expected to develop tissue-like higher order intercellular structures (cell-cell contacts, extracellular matrix) that orchestrate cellular functions including proliferation, differentiation, apoptosis, and angiogenesis with unmatched quality. We have refined the hanging drop cultivation technology to pioneer beating heart microtissues derived from pure primary rat and mouse cardiomyocyte cultures as well as mixed populations reflecting the cell type composition of rodent hearts. Phenotypic characterization combined with detailed analysis of muscle-specific cell traits, extracellular matrix components, as well as endogenous vascular endothelial growth factor (VEGF) expression profiles of heart microtissues revealed (1) a linear cell number-microtissue size correlation, (2) intermicrotissue superstructures, (3) retention of key cardiomyocyte-specific cell qualities, (4) a sophisticated extracellular matrix, and (5) a high degree of self-organization exemplified by the tendency of muscle structures to assemble at the periphery of these myocardial spheroids. Furthermore (6), myocardial spheroids support endogenous VEGF expression in a size-dependent manner that will likely promote vascularization of heart microtissues produced from defined cell mixtures as well as support connection to the host vascular system after implantation. As cardiomyocytes are known to be refractory to current transfection technologies we have designed lentivirus-based transduction strategies to lead the way for genetic engineering of myocardial microtissues in a clinical setting.

Identification of a Golgi-localised GRIP domain protein from Arabidopsis thaliana

A family of Golgi-localised molecules was recently described in animals and fungi possessing extensive coiled regions and a short (similar to40 residues) conserved C-terminal domain, called the GRIP domain, which is responsible for their location to this organelle. Using the model plant Arabidopsis thaliana, we identified a gene (AtGRIP) encoding a putative GRIP protein. We demonstrated that the C-terminal domain from AtGRIP functions as a Golgi-targeting sequence in plant cells. Localisation studies in living cells expressing the AtGRIP fused to a DsRed2 fluorescent probe, showed extensive co-location with the Golgi marker alpha-mannosidase I in transformed tobacco protoplasts. GRIP-like sequences were also found in genomic databases of rice, maize, wheat and alfalfa, suggesting that this domain may be a useful Golgi marker for immunolocalisation studies. Despite low sequence identity amongst GRIP domains, the plant GRIP sequence was able to target to the Golgi of mammalian cells. Taken together, these data indicate that GRIP domain proteins might be implicated in a targeting mechanism that is conserved amongst eukaryotes.

Post-transcriptional regulation of the cystic fibrosis gene in cardiac development and hypertrophy

Eukaryotic gene expression, reflected in the amount of steady-state mRNA, is regulated at the post-transcriptional level. The 5'-untranslated regions (5'-UTRs) of some transcripts contain cis-acting elements, including upstream open reading frames (uORFs), that have been identified as being fundamental in modulating translation efficiency and mRNA stability. Previously, we demonstrated that uORFs present in the 5'-UTR of cystic fibrosis transmembrane conductance regular (CFTR) transcripts expressed in the heart were able to modulate translation efficiency of the main CFTR ORF. Here, we show that the same 5'-UTR elements are associated with the differential stability of the 5'-UTR compared to the main coding region of CFTR transcripts. Furthermore, these post-transcriptional mechanisms are important factors governing regulated CFTR expression in the heart, in response to developmental and pathophysiological stimuli. (C) 2004 Elsevier Inc. All rights reserved.

Arp2/3 activity is necessary for efficient formation of E-cadherin adhesive contacts

Classical cadherin adhesion molecules are fundamental determinants of cell-cell recognition that function in cooperation with the actin cytoskeleton. Productive cadherin-based cell recognition is characterized by a distinct morphological process of contact zone extension, where limited initial points of adhesion are progressively expanded into broad zones of contact. We recently demonstrated that E-cadherin ligation recruits the Arp2/3 actin nucleator complex to the plasma membrane in regions where cell contacts are undergoing protrusion and extension. This suggested that Arp2/3 might generate the protrusive forces necessary for cell surfaces to extend upon one another during contact assembly. We tested this hypothesis in mammalian cells by exogenously expressing the CA region of N-WASP. This fragment, which potently inhibits Arp2/3-mediated actin assembly in vitro, also effectively reduced actin assembly at cadherin adhesive contacts. Blocking Arp2/3 activity by this strategy profoundly reduced the ability of cells to extend cadherin adhesive contacts but did not affect cell adhesiveness. These findings demonstrate that Arp2/3 activity is necessary for cells to efficiently extend and assemble cadherin-based adhesive contacts.

Clathrin-independent endocytosis: New insights into caveolae and non-caveolar lipid raft carriers

A number of recent studies have provided new insights into the complexity of the endocytic pathways originating at the plasma membrane of mammalian cells. Many of the molecules involved in clathrin coated pit internalization are now well understood but other pathways are less well defined. Caveolae appear to represent a low capacity but highly regulated pathway in a restricted set of tissues in vivo. A third pathway, which is both clathrin- and caveolae-independent, may constitute a specialized high capacity endocytic pathway for lipids and fluid. The relationship of this pathway, if any, to macropinocytosis or to the endocytic pathways of lower eukaryotes remains an interesting open question. Our understanding of the regulatory mechanisms and molecular components involved in this pathway are at a relatively primitive stage. In this review, we will consider some of the characteristics of different endocytic pathways in high and lower eukaryotes and consider some of the common themes in endocytosis. One theme which becomes apparent from comparison of these pathways is that apparently different pathways can share common molecular machinery and that pathways considered to be distinct actually represent similar basic pathways to which additional levels of regulatory complexity have been added. (c) 2005 Elsevier B.V. All rights reserved.

Gene codon composition determines differentiation-dependent expression of a viral capsid gene in keratinocytes in vitro and in vivo

By establishing mouse primary keratinocytes (KCs) in culture, we were able, for the first time, to express papillomavirus major capsid (L1) proteins by transient transfection of authentic or codon-modified L1 gene expression plasmids. We demonstrate in vitro and in vivo that gene codon composition is in part responsible for differentiation-dependent expression of L1 protein in KCs. L1 mRNA was present in similar amounts in differentiated and undifferentiated KCs transfected with authentic or codon-modified L1 genes and had a similar half-life, demonstrating that L1 protein production is posttranscriptionally regulated. We demonstrate further that KCs substantially change their tRNA profiles upon differentiation. Aminoacyl-tRNAs from differentiated KCs but not undifferentiated KCs enhanced the translation of authentic L1 mRNA, suggesting that differentiation-associated change to tRNA profiles enhances L1 expression in differentiated KCs. Thus, our data reveal a novel mechanism for regulation of gene expression utilized by a virus to direct viral capsid protein expression to the site of virion assembly in mature KCs. Analysis of two structural proteins of KCs, involucrin and keratin 14, suggests that translation of their mRNAs is also regulated, in association with KC differentiation in vitro, by a similar mechanism