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Establishment and characterization of Prnp knockdown neuroblastoma cells using dual microRNA-mediated RNA interference

Establishment and characterization of Prnp knockdown neuroblastoma cells using dual... RESEARCH PAPER RESEARCH PAPER Prion 5:2, 93-102; April/May/June 2011; © 2011 Landes Bioscience Establishment and characterization of Prnp knockdown neuroblastoma cells using dual microRNA-mediated RNA interference 1,2,† 1,† 2 2 2 2 3 Sang-Gyun Kang, Yu-Mi Roh, Agnes Lau, David Westaway, Debbie McKenzie, Judd Aiken, Yong-Sun Kim 1, and Han Sang Yoo * Department of Infectious Diseases; College of Veterinary Medicine; KRF Zoonotic Disease Priority Research Institute and BK21 Program for Veterinary Science; 2 3 Seoul National University; Seoul, Korea; Centre for Prions and Protein Folding Diseases; University of Alberta; Edmonton, AB Canada; Ilsong Institute of Life Science and Department of Microbiology; College of Medicine; Hallym University; Anyang, Korea These authors contributed equally to this work. Key words: N2a, miRNA, PrP , Prnp, RNAi Prion diseases are fatal transmissible neurodegenerative disorders. In the pathogenesis of the disease, the cellular prion C Sc Sc protein (PrP ) is required for replication of abnormal prion (PrP ), which results in accumulation of PrP . Although there have been extensive studies using Prnp knockout systems, the normal function of PrP remains ambiguous. Compared with conventional germline knockout technologies and transient naked siRNA-dependent knockdown systems, newly constructed durable chained-miRNA could provide a cell culture model that is closer to the disease status and easier to achieve with no detrimental sequelae. The selective silencing of a target gene by RNA interference (RNAi) is a powerful approach to investigate the unknown function of genes in vitro and in vivo. To reduce PrP expression, a novel dual targeting-microRNA (miRdual) was constructed. The miRdual, which targets N- and C-termini of Prnp simultaneously, more ©2011 Landes Bioscience. effectively suppressed PrP expression compared with conventional single site targeting. Furthermore, to investigate the C C cellular change following PrP depletion, gene expression analysis of PrP interacting and/or associating genes and several assays including proliferation, viability and apoptosis were performed. The transcripts 670460F02Rik and Plk3, Ppp2r2b Do not distribute. and Csnk2a1 increase in abundance and are reported to be involved in cell proliferation and mitochondrial-mediated apoptosis. Dual-targeting RNAi with miRdual against Prnp will be useful for analyzing the physiological function of PrP in neuronal cell lines and may provide a potential therapeutic intervention for prion diseases in the future. C 8,9 physiological role of PrP Introduction . These transgenic animal models are, 10,11 however, expensive and time consuming. To overcome these Prion diseases are group of fatal transmissible neurodegenerative problems, cell culture models of prion diseases have been used for disorders characterized by a long incubation period and broad screening and elucidating the mechanism of action of anti-prion neuropathology including spongiform encephalopathy, glio- agents and to analyze biological properties of PrP at the molecu- 1 7,11,12 sis and neuronal loss. They are classified into sporadic, inher - lar and cellular levels. Elucidating the cellular function of ited and acquired forms, which can spread within and between PrP may help in deciphering mechanisms of prion pathogenesis 2 5,13 mammalian species. The central feature of prion diseases is the and in devising therapeutic strategies. C C conversion of a host-encoded prion protein (PrP ) into a dis- Mature PrP translocates to the outer leaflet of the plasma Sc ease-associated isoform (PrP ), which shares same amino acid membrane in proximity to raft-associated signaling molecules. sequence with PrP but is posttranslationally converted to the It traffics in and out of lipid rafts and is involved in a diversity 14-16 infectious form, accumulating primarily in the central nerve sys- of molecular and physiological functions. These proper- 3 C C tem. PrP is implicated in prion pathogenesis as it is required for ties of PrP suggest the possibility that several changes in Prnp propagation and development of prion pathology. Although loss knockout cell culture models may be caused by an altered expres- C Sc of PrP function or deposition of PrP is not sufficient to cause sion of interlinked genes with different cellular functions. Prnp Sc the disease, the process of conversion into PrP may alter signal knockouts could reflect not only the missing function but also 5-7 transduction, thereby giving a toxic dominant function. the compensation for that missing function, as gene deletions in There have been extensive efforts to develop prion disease individual cells or tissues are often accompanied by compensa- 17,18 models in vivo, especially Prnp knockout models for defining the tory changes in gene expression patterns. *Correspondence to: Han Sang Yoo; Email: [email protected] Submitted: 09/20/10; Accepted: 03/28/11 DOI: 10.4161/pri.5.2.15621 www.landesbioscience.com Prion 93 Results Establishment of stable Prnp knockdown cells. Knockdown of a gene using exogenously introduced siRNA is always transient due to a cell division and/or degradation of siR NA molecules. To develop stable Prnp knockdown cells, small hairpin struc- tural artificial miRs were designed ( Fig. 1A). The direction and sequence of the artificial miRs were confirmed by sequence analysis, and N2a cells were transfected with the either miR1, miR2, miRdual or miRscr constructs. The transient effects of each miR on prion protein expression were assessed by western blot analysis. The miRdual most effectively knocked down the expression of PrP by 75 ± 2% compared with wild-type N2a cells (p < 0.01), whereas PrP level in N2a cells transfected with miR1, miR2 and miRscr was decreased by 17 ± 7%, 24 ± 9% (p < 0.05) and no knockdown effect, respectively (Fig. 1B and C). To establish stable Prnp knockdown cells, the N2a cells transfected with miRdual or miRscr were selected with Blasticidin S for a month and are referred to here as N2amiRdua l and N2amiRscr, respectively. The single clones were further selected by limiting dilution and clonally expanded cells were analyzed by genomic PCR, qPCR and western blot to confirm stable transformation with miR as well as transcription and expression of Prnp. Association of the miR plasmid with host chromosomal sequences was assessed by PCR of genomic DNA. ©2011 Landes N2 amB iRdi uao l tes sted pc oi site ive fn or tc he 5e 50 b. p amplicon, while Figure 1. Schematic features of miR cassettes and knockdown efficacy N2amiRscr yielded 412 bp amplicon. No amplicon occurred in N2a cells. (A) Each miR cassette in pcDNA6.2-GW/EmGFP vector. with genomic DNA from wild-type N2a cells (Fig. 2A). To (B) Western blot of N2a cells transfected with each miR expression construct. (C) Quantitation of PrP expression. When miRdual was intro- quantify the effect of miR on Prnp transcription, qPCR was Do not distribute. duced into the cells, PrP expression was the most efficiently decreased used and transcription input of each sample was normalized as compared with wild-type cells (*p < 0.05, **P <0.01). using qPCR of Gapdh. The Prnp transcript was decreased by 87 ± 1% in N2amiRdual (p < 0.01), while it was 50 ± 15% RNA interference (RNAi) refers to the use of 21–23 nucleo- lower in N2amiRscr compared with wild-type N2a (p < 0.05) tide short interfering R NA s (siR NA s) mediating post-transcrip- (Fig. 2B). PrP expression was quantified by western blot. Band tional degradation or translational repression of homologous intensity analysis of western blot indicated that the resultant gene transcripts. Stable knockdowns can be obtained by consti- protein expression of PrP was decreased in N2amiRdual by 96 19,20 tutive expression of the siRNA from the host chromosome. ± 1% and 94 ± 2% in two different cell clones compared with RNAi has been accepted as the most powerful reverse-genetics wild-type (p < 0.01). N2amiRscr showed a reduced expression approach in mammalian cells, however, there are significant of PrP by 27 ± 4% (p < 0.05) (Fig. 2C). technical limitations including identifying efficient methods to Differential gene expression in stably transformed design and deliver siRNA. Unlike other approaches, such as N2amiRdual cells. We next analyzed gene expression changes traditional gene targeting by homologous recombination, anti- in response to Prnp knockdown. These studies were undertaken sense vectors and catalytic RNA or DNA molecules, RNAi is in N2a cells and N2amiRdual derivative and corresponded an endogenous natural pathway and allows cross-species appli- to the quantified expression of several target genes thought to 12 C cation. RNAi, which acutely decreases target expression, also interact and/or associate with PrP (Table 1). For all targets, has the advantage of avoiding compensatory or mechanistic transcript inputs were normalized using Gapdh and standard adaptations. curves were generated using serially diluted PCR products of The present study describes the development of a prion each specific gene. Relative expression levels were expressed as knockdown cell culture model by introducing artificial percent compared to wild-type N2a cells. Of the seven targets microR NA (miR) targeting Prnp into the mouse neuroblastoma selected, 670460F02Rik, Csnk2a1, Plk3 and Ppp2r2b increased cell line (N2a) as well as RK13 cells expressing mouse PrP . in abundance (220 ± 33%, 230 ± 52%, 183 ± 10% and The established Prnp knock-down N2a cells were characterized 519 ± 5%, respectively) in N2amiRdual cells compared with by investigating the expression profiles of the genes known as wild-type N2a cells (p < 0.01) (Fig. 3). The abundance of these PrP interacting and/or associating molecules, proliferation, transcripts also increased, but to a less degree, in N2amiRscr viability and apoptotic resistance. (121 ± 6%, 132 ± 11%, 147 ± 16% and 350 ± 21% respectively). 94 Prion Volume 5 Issue 2 The trend in expression patterns for all target genes was negatively correlated with the amount of PrP in N2amiRdual and N2amiRscr cells (Fig. 2B and C). For Mpg, the mRNA expression was increased both in N2amiRdual and N2amiRscr by 195 ± 21% and 184 ± 24%, respectively (p < 0.05) (Fig. 3). As expected, the Cdr34 and Gfap transcripts as controls were not detectable until 40 cycles of qPCR reac- tion in both N2amiRs and wild-type N2a cells. Alteration in cellular characteristics following PrP k nock-down. A fter estab- lishment of the prion knockdown cell line, N2amiRdual, proliferation, viability and mitochondrial-mediated apoptosis were examined. In proliferation assays, Figure 2. Transfection of miR cassettes and Prnp knockdown efficacy at mRNA and protein level. the N2amiRdual showed decreased pro- (A) PCR of genomic DNA shows miR cassette-specific amplicons. Lane L, 100 bp DNA ladder; lane 1, expression vector with miRdual cassette; lane 2, expression vector with miRscr; lane 3, wild- liferation, to 88 ± 4%, in mitochondrial type cells; lane 4, N2amiRdual; lane 5, N2amiRscr; lane 6, no template. (B) qPCR analysis of PrP dehydrogenase activity tests compared transcript abundance showing knockdown efficiency of miRdual in mRNA levels. (C) Western blot with wild-type (p < 0.05). No significant analysis of PrP protein abundance. Top part is the western blot and bottom panel is densitomet- difference was observed in lactate dehy- ric evaluation. dual1 and dual2 are N2amiRdual cell clones (*p < 0.05, **p < 0.01). drogenase tests for cell viability (Fig. 4A). Based on the observed morphology of the cell body an© d neuri2 tis, n0 o d1 iffe1 renc eL s wea re on bserd ved ie n s (Fi g.B 5C). Tio he ps ropoc rtioi n oe f apn optoc tic ce ells b. ound to Annexin V N2amiRdual and N2amiRscr compared with wild-type. Both was increased by 212 ± 4% in N2amiRdual at 24 h post apoptosis cell types expressed the GFP reporter molecules, however, the induction when compared to the percentage of dying cells before signal was stronger in miRscr cassette than in miRdual (Fig. apoptosis induction (p < 0.05). Most N2amiRscr and wild-type Do not distribute. 4B). GFP signals derived from miR expression cassette were N2a cells were resistant to apoptotic stimulation regardless of attenuated in N2amiRdual cells, likely due to increased pro- growth environment (Fig. 6). cessing of the miRdual. Lentiviral transduction of RK13 expressing mouse PrP To measure mitochondrial-mediated apoptosis induced by with miRs. To confirm the reduction of PrP levels by the dual serum withdrawal, the expression level of mitochondria fission knockdown method, a stable clone of rabbit kidney epithelial 23 C related protein, dynamin related protein 1 (Drp1), was quanti- cells (RK13), expressing both mouse PrP (RK13moPrnp) and fied. Drp1 expression was increased in N2amiRdual by 112 ± GFP was transduced with lentivirus containing either the miR- 4% 24 h following apoptosis induction when compared to the dual or miRscr expression cassettes. The cells were harvested at level before apoptosis induction (p < 0.05). There was, however, 70% and 100% conu fl ence and the relative abundance of PrP no significant difference in the Drp1 expression level between analyzed by western blot (Fig. 7A). In these analyses quanti- N2amiRscr and N2a wild-type, regardless of growth condition ties of PrP and GFP were normalized to the sample with either such as serum deprivation (Fig. 5A). The level of pro-apop- the miRdual or miRscr at 70% cell conu fl ency. PrP level was totic Cytochrome C (Cyt c) in cytosol increased 215 ± 2% in decreased in RK13moPrnp cells with miRdual (46 ± 8% and N2amiRdual cells during serum deprivation compared to the 58 ± 5%), while no significant difference in PrP level was level before serum deprivation (p < 0.05). Again there was no observed in RK13moPrnp cells with miRscr (p < 0.05) (Fig. 7B). difference between N2amiRscr and wild-type N2a cells even Besides assessing protein loading with an actin probe, the GFP though cells had undergone apoptotic stimulation (Fig. 5B). also encoded within the bigenic plasmid was evaluated by use of During apoptotic stimulation, morphological changes between an anti-GFP antibody. No diminution was seen when using the each cell lines were observed by light microscopy. Neurite retrac- “dual” lentivirus, speaking to specificity, and in fact GFP protein tion was induced and proliferation inhibited in N2amiRdual levels were elevated by ~30% both in RK13moPrnp-miRdual cell 24 h post serum deprivation, while N2amiRscr and wild- (136 ± 4% and 139 ± 7%) and in RK13moPrnp-miRscr cells type N2a cells maintained their structural integrity. The caspase (127 ± 6% and 129 ± 4%). This effect cannot represent alle- 3 activity in N2amiRdual cells was increased from 1.6 pmol viation of a transcription interference effect from Prnp cassette pNA liberated/hour at 0 h to 18.8 pmol pNA liberated/hour at within the bigenic pBUD vector (which has tandem expression 48 h post serum deprivation (p < 0.01), whereas there were no cassettes), as it is seen with viruses with different effects upon significant differences in N2amiRscr and wild-type N2a cells PrP expression. www.landesbioscience.com Prion 95 Table 1. Target genes of qPCR for characterizing the established N2amiRdual cell line Gene Gene Name GenBank Accession No. General Putative Function Ref. Symbol C C The function of PrP is not known. PrP is encoded in the host Prnp prion protein NM_011170 genome and is expressed both in normal and infected cells. RIKEN cDNA 6720460F02 gene AK049710 Unknown 30 F02Rik In fission yeast, acts as a mitotic inducer. In G it negatively regu- Cerebellar degeneration- Cdr34 NM_001166658 lates wee1, a mitotic inhibitor. Also has a role in cytokinesis where it 31 related antigen 1 required for proper septum formation. Casein kinases are operationally defined by their preferential utiliza- casein kinase 2, alpha1 Csnk2a1 AK011501 tion of acidic proteins such as caseins as substrates. The alpha and 32 polypeptide alpha’ chains contain the catalytic site. GFAP, a class-III intermediate filament, is a cell-specific marker that, Gfap Glial fibrillary acidic protein AF332061 during the development of the central nervous system, distinguishes 33 astrocytes from other glial cells. Hydrolysis of the deoxyribose N-glycosidic bond to excise 3-meth- N-methylpurine-DNA Mpg NM_010822 yladenine, and 7-methylguanine from the damaged DNA polymer 30 glycosylase formed by alkylation lesions. Serine/threonine protein kinase involved in regulating M phase Plk3 Polo-like kinase 3 (Drosophila) NM_013807 functions during the cell cycle. May also be part of the signaling net- 30 work controlling cellular adhesion. pro© tein pho2 spha0 tase 2 1 1 Landes The B r B egulai toro y subs unit mc igi ht me odn ulate sc ube strate s. electivity and Ppp2r2b (formerly 2A), regulatory sub- NM_028392 catalytic activity, and also might direct the localization of the cata- 31 unit B (PR 52), beta isoform lytic enzyme to a particular subcellular compartment. Do not distribute. Figure 3. Quantitative PCR analysis of PrP interacting and/or associ- ated molecules. 6720460F02Rik, Plk3, Ppp2r2b, Csnk2a1 transcripts were upregulated in prion knockdown N2amiRdual cells compared to wild- type N2a cells. The increased Mpg transcripts in both N2amiRdual and N2amiRscr seemed to have no correlation with decreased PrP (*p < 0.05, **p < 0.01). Discussion The knockdown of PrP in N2a cells using a miR-based RNAi system provides a novel cell culture model for studying prion biology. The artificial miRs were designed to target nucleo - tide residues 36–56 and 668–688 of mouse PrP ORF region, respectively. The miRs were constructed to be expressed as a cluster in a long primary transcript driven by RNA polymerase II. This dual targeting strategy enhanced knockdown efficacy more than 3-fold compared to single site targeting method. It was reported that a single miR can bind to and regulates many different mRNA target, conversely, several different miRs can 25,26 pair with and cooperatively control a single mRNA target. Downregulation of Prnp was also observed in N2a transfected with miRscr, an unexpected result as the scrambled sequence does not target any known vertebrate gene. To better define this targeting effect of miRscr to Prnp, we examined RK13moPrnp cells, which were lentiviral-transduced with miRdual or miRscr, respectively. In the viral-tranduced cells with miRdual, the expression levels of PrP varied inversely with the amount of 96 Prion Volume 5 Issue 2 GFP. These results suggest that, rather than specific targeting by miRscr, the reduced PrP expression in N2amiRscr is likely due to exogenous factors through gene transfection, such as genomic 27-29 insertion of foreign genes that may disrupt cellular processes. An exploration of cellular interactors is one of the way to disclose unknown function of protein of interest. Transcript abundances of PrP interacting and/or associating proteins fol- lowing stable Prnp knockdown can provide valuable information concerned with normal cellular function of PrP . Therefore, we selected seven target genes from previously reported microarray 30-33 and protein microarray data and transcript abundance was investigated by qPCR. Of the seven genes examined in this study, the abundance of four PrP interactors increased following Prnp downregulation. One of the upregulated genes in N2amiRdual is 6720460F02Rik gene, also known as CATS (computer-annotated as FAM64A), the Homo sapiens family with sequence similarity 64, member A in GenBank (NM_144526). Its transcript is abun- dantly expressed in proliferative state brain tissues, glioma and Figure 4. Cell proliferation, viability and GFP expression test. (A) Viability primitive neuroectodermal tumors, however, the precise cellular and proliferation of N2a cells stably transformed with miRdual or miRscr function of the protein is unknown. FAM64A was shown to (*p < 0.05). (B) Confocal microscopy images of clonal expanded cells. interact with PrP through protein microarray and coimmuno- N2amiRdual and N2amiRscr showed green fluorescent signals. Cell nuclei were stained with DAPI (blue). Photos were taken at 20 doubling passages. precipitation with recombinant human PrP spanning amino acid GFP signals stably expressed over 135 doubling passages. Scale bar, 50 um. residues 23–231. In addition, FAM64A is located predominantly in the nucleus, where it colocalizes with the recombinant human C 30 PrP . This suggests that FAM64A/CATS/6720460F02Rik might have a role in c© ontro2 l of c0 ell p1 rol1 ifer atL ion ca onsn istend t wie th s Bioscience. the decreased cell proliferation observed in our studies. The increased transcription level could be a compensatory reaction to the loss of PrP function associated with cell proliferation. Do not distribute. Plk3 is a member of the polo family of serine/threonine kinases and is also as a PrP interacting partner. It localizes to the nucleolus and is involved in regulation of the G /S phase tran- 35,36 C sition. The control of cell proliferation by PrP may alter the expression of cell cycle-related genes, such as cyclin D1, Esp8 and CD44, in a cell type-specific way. The increased level of Plk3 in N2amiRdual cells may be a compensatory response to a PrP - dependent cell proliferation event. Moreover, the overexpression of Plk3 may mediate inhibition of proliferation as a result of the C 36,37 loss of the anti-apoptotic function of PrP . Further analysis into the mechanism of PrP function associated with Plk3 will be required. Ppp2r2b encodes the neuron-specific regulatory beta subunit of the protein phosphatase 2A (PP2A) holoenzyme and the muta- tions in Ppp2r2b are responsible for the neurodegenerative disor- der, spinocerebellar ataxia 12. Increased expression of Ppp2r2b reported to induce mitochondrial fragmentation through stimu- lating mitochondrial fission protein such as Drp1, which can lead 38,39 to neuronal apoptosis. Our observation of increased expres- sion of Ppp2r2b following PrP knockdown was not consistent Figure 5. Apoptotic resistance to serum deprivation. Cells were cultured under conditions of serum deprivation and collected at indicated time points. (A) Detection of Drp1 expression levels analyzed by densitometric analysis of western blot. (B) Cytochrome C expression in cytosolic frac- tions detected by immunoassay. (C) Analysis of caspase 3 activity calcu- lated by comparison with the free pNA. dual, N2amiRdual; scr, N2amiRscr; wt, miR non-treated wild-type N2a (*p < 0.05, **p < 0.01). www.landesbioscience.com Prion 97 ©2011 Landes Bioscience. Do not distribute. Figure 6. Detection of apoptotic cells following serum deprivation using Annexin V-Cy3 assay. Cells were collected at the indicated time points and 5 x 10 cell suspensions were treated with Annexin V. The proportion of cells showing apoptotic change was increased in N2amiRdual whereas most of cells showed the resistance against apoptosis stimulation in N2amiRscr and wild-type N2a cells (*p < 0.05). C 43,44 with previous study that showed the increased Ppp2r2b in a PrP by environmental genotoxins. The upregulation of Mpg both overexpressing HEK293 cell line. It is conceivable that dysreg- in N2amiRdual and N2amiRscr is curious, but may be a response ulation of Ppp2r2b via abnormal expression of PrP might be a to the expression of double-stranded miR that has internal loops. critical cause of cell death secondary to mitochondrial dysfunction. There was no difference observed in viability of the N2a lines Mitochondrial fragmentation by overexpression of Ppp2r2b occurs but proliferation was decreased in N2amiRdual compared with upstream of apoptosis and was sufc fi ient for hippocampal neuron N2amiRscr and wild-type cells. This is likely due to the down- 39 C death. It is consistent that transfection of PrP knockout cells with regulation of PrP rather than other exogenous factors. It has 37 C a Prnp expression vector prevented typical apoptotic changes. been shown that the expression level of PrP was positively cor- 37,45 Casein kinase 2 (CK2) participates in the regulation of diverse related with the cell proliferation. In agreement with other 46-48 cellular processes. It is a messenger-independent protein serine/ studies of prion knockout model, newly constructed prion threonine kinase and especially abundant in the brain. It is local- knockdown N2amiRdual cells were more vulnerable to apop- C 40 ized to the outer plasma membrane where PrP also resides. The totic stimulation than N2amiRscr and wild-type N2a. Inhibition catalytic α subunit of CK2 (CK2α), encoded by the Csnk2a1 gene, of Drp1 activity during apoptosis in mammalian cells inhibits C 41 is reported to bind to cytosolic non-glycosylated PrP . CK2α mitochondrial fragmentation, Cyt c release and the rate of cell 39,49,50 also binds to PP2A in mitogen-starved cells in vitro and overex- death. Altering the mitochondrial fusion/fission balance pression of CK2α results in suppression of cell growth. These towards fission by Drp1 could lead to cell death by promoting reports are consistent with the result of decreased proliferation mitochondrial release of pro-apoptotic proteins including Cyt c. in N2amiRdual cells that may cause compensatory increases of The release of Cyt c from mitochondrial inter-membrane space C 51,52 Ppp2r2b and Csnk2a1 following decreased expression level of PrP . to the cytosol has reported to activate the caspase family, an The Mpg gene encodes the base-excision repair enzyme, important event for the downstream activation of apoptosis cas- 49,53 3-methyladenine DNA glycosylase and has been reported as a cade. This phenomenon is supported by Cyt c knockout cell PrP interacting protein. Mpg plays a vital role in preserving cer- lines study showing reduced caspase 3 activation and resistance ebellar development and protecting mature neurons from insults to various apoptotic stimuli. 98 Prion Volume 5 Issue 2 In conclusion, the stable N2amiRdual cells will be a useful tool for elucidating the physiological function of PrP and neu- ropathogenesis of prion disease. Moreover, since miR is known as more suitable approach for achieving RNAi in mouse brain, in terms of toxicity, it is expected that the dual targeting artifi - cial miR-based RNAi used in this study will provide a promising therapeutic intervention for prion diseases and other neurodegen- erative disorders. Materials and Methods Cell culture. N2a and RK13moPrnp cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) with high glu - cose (4.5 g/l) and 2 mM glutamine (Gibco , Invitrogen, #11995), supplemented with 10% fetal bovine serum, penicillin and strep- tomycin. Cells were cultured at 37°C in a 5% CO incubator. Designing artificial miRNAs. miRs were designed to knock- down the endogenous Mus musculus Prnp in N2a cells based on previous report in reference 56. The miR design algorithm, provided by the Invitrogen web-site, was used to select target sequences. The basic local alignment search tool (BLAST) was used with the mouse genome database to identify unique regions in the murine Prnp open reading frame (ORF, NM_011170). Two regions were chosen to target; the N-(miR1) and C-termini (miR2) of the Prnp ORF. miR1 and miR2 bind at 191 to 211 and 823 to 843 of Pr© np O2 RF re0 gio1 n, re1 sp ecL tivea ly. En ach sid ngle e-s Bioscience. stranded DNA (ssDNA) was designed to contain the antisense C Figure 7. Downregulation pattern of PrP in RK13-moPrnp cells sequence derived from target sites, followed by 19 nucleotides to harvested at different confluences. RK13-mo Prnp cells were lentiviral- transduced with miR expression cassettes and the relative abundances form the terminal loop and sense target sequence with 2 nucleo- Do not distribute. of PrP and GFP processed were determined by western blot (*p < 0.05, tides removed to create an internal loop (Table 2). **p < 0.01). Construction and expression of miRNAs. The miRs were synthesized commercially (Invitrogen) and annealed for direc- tional cloning. The synthesized top- and bottom-strand DNAs and analyzed by expression of green fluorescent protein (GFP) (Table 2) were annealed and ligated into pcDNA6.2-GW/ reporter gene, genomic PCR, quantitative PCR (qPCR) and EmGFP-miR (Invitrogen, #K4936-00) expression vector. The western blot. ligation mixture was transformed into TOP10 competent E. coli. Transfection of RK13 cells with mouse Prnp. RK13 cells To confirm the orientation and DNA sequence of the miRs, each were transfected with a derivative of the bigenic pBud mamma- construct was sequenced using an automated DNA Sequencer lian vector expressing both GFP and a wt mouse Prnp coding (ABI PRISM 377 L). To express miRs as one primary transcript, region. This procedure used lipofectamine2000 (Invitrogen, miR2 cassette were digested and ligated into miR1 construct. #11668). Cells stably expressing mouse PrP (RK13moPrnp) The linked miRs (miRdual) was transformed and analyzed as were selected with Zeocin (Invitrogen, #R250) and screened by described above. A miR cassette containing a scramble sequence western blot. (miRscr) (Invitrogen, #K4936-00), which can be processed into Production of lentiv ir us a nd infection of R K13moPrnp. The mature miR but is not predicted to target any known vertebrate m i R e x pre s sion c a s set te s i nc lud i ng GFP were c loned i nto pL ent i6 / gene, was used as a negative control (Fig. 1A). V5-DEST lentiviral vectors (Invitrogen, #K4937) by Gateway Transfection of N2a cells with miRNAs. The wild-type N2a site-specific recombination. The constructed lentiviral vectors cell line was transfected with the expression construct using the (3 μg) were co-transfected with a third-generation lentivirus FuGENE6 transfection reagent (Roche, #11814443001). At 24 h packaging vector (9 μg, Invitrogen, # K4970) into HEK293FT post transfection, the cells were lysed with RIPA lysis buffer cells using Lipofectamine 2000 reagent (Invitrogen, #11668). (1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, The viral supernatant was collected 48 h after co-transfection 50 mM Tris-HCl, pH 7.4, 0.1% SDS, 1 mM EDTA) contain- and concentrated by ultracentrifugation for 2 h at 153,725 g and ing a protease inhibitor cocktail (Amresco, #M222) and the 4°C. The titer of lentiviruses was determined using HT1080 efficiency of downregulation of Prnp was measured by western cells and RK13moPrnp cells were infected with the virus par- blot. Through drug selection with Blasticidin S (Invitrogen, ticles at an MOI of 10. After Blasticidin S selection for two #R 21001) and limiting dilution, clonal expanded cells expressing weeks, cells were analyzed for knockdown of PrP by western miRdual (N2amiRdual) or miRscr (N2amiRscr) were obtained blot. www.landesbioscience.com Prion 99 Table 2. Sequences of artificial miRs targeting Mus musculus Prnp ORF Sequence L Mature miRNA Sequence Loop Sequence Sense Sequence L T TGC TG ACA TCA GTC CAC ATA GTC ACA GTT TTG GCC ACT GAC TGA C TGT GAC TAT GGA CTG ATG T miR1 B C TGT AGT CAG GTG TAT CAG TGT CAA AAC CGG TGA CTG ACT G ACA CTG ATA CCT GAC TAC A GTC C T TGC TG ATC TTC TCC CGT CGT AAT AGG GTT TTG GCC ACT GAC TGA C CCT ATT ACC GGG AGA AGA T miR2 B C TAG AAG AGG GCA GCA TTA TCC CAA AAC CGG TGA CTG ACT G GGA TAA TGG CCC TCT TCT A GTC C T TGC TG AAA TGT ACT GCG CGT GGA GAC GTT TTG GCC ACT GAC TGA C GTC TCC ACG CAG TAC ATT T miRscr B C TTT ACA TGA CGC GCA CCT CTG CAA AAC CGG TGA CTG ACT G CAG AGG TGC GTC ATG TAA A GTC C L, linker; T, top strand; B, bottom strand. Genomic PCR. Genomic DNA of clonal expanded cells was iso- for the ABI PRISM 7300 (Applied Biosystems) was performed lated using a genomic DNA puric fi ation kit (Promega, #A11120). as follows: 2 min at 50°C for uracil N-glycosylase (UDG) incu- Primer pairs were designed to amplify specic fi miR cassettes bation which removes uracil-containing products from previous (Table 3). The PCR parameters consisted of an initial denatur- reactions, UDG inactivation and DNA polymerase activation for ation step at 95°C for 5 min followed by 40 cycles of denaturation 2 min at 95°C, followed by 40 cycles of 15 sec at 95°C for dena- at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at turation and 30 sec at 60°C for hybridization and elongation. 72°C for 1 min and a final extension at 72°C for 15 min. PCR Proliferation and viability assay. The established N2a- products were analyzed by electrophoresis on 1.0% agarose gel. miRdual, N2amiRscr and wild-type cell lines were cultured in Western blot. For western blot analysis, 5 μg of pro- 96-well plates with 3 x 10 cells in 100 μl of DMEM culture tein from each cell lysate was prepared and fractionated on a medium per each triplicate well. Activity of cellular mitochon- 12% Tris-glycine gel and transferred to a 0.2 micron nitro- drial and lactate dehydrogenase were determined using commer- cellulose membrane using a dry blotting system (Invitrogen, cial kits (Biovision, #302-500, #313-500) to measure proliferation #IB1001). The mem© brane w2 a0 s blo1 cke1 d w iL th 5a % nn onfd at de ry s and c B ells vi iao bilits y rec speci tie vely. An ftec r 4e 8 h i. ncubation, the tetra- milk in TritonX-Tris buffered saline (TTBS: 0.1% TritonX-100 zolium salt (WST) was added to each well and the amount of in 100 mM Tris-HCl, pH 7.5, 0.9% NaCl) and then probed formazan production quantified using a microtiter plate reader at with anti-PrP antibody, 3F10, anti-GFP antibody (AbCAM, 450 nm. Non-viable controls were prepared by treating cells with Do not distribute. #ab290) or anti-dynamin-related protein 1 (Drp1) antibody 1% TritonX-100. (BD Transduction Laboratories, #611112) at 4°C for over- Cytochrome C assay. The cytosolic and mitochondrial frac- night. An anti-mouse IgG conjugated to horseradish peroxi- tions from each cell line were separated using a mitochondria iso- dase (Santa Cruz Biotechnology, #SC-2005) was used as the lation kit (Pierce, #89874) following manufacturer’s instruction. secondary antibody. The bound antibodies were visualized by Total protein concentration of the cy tosolic fractions were adjusted chemiluminescence (Amersham, #RPN2106) and protein lev- using BCA protein assay (Pierce, #23227) and the amount of Cyt els were measured by scanning and evaluating density (Multi c in each fraction were analyzed by sandwich Enzyme Linked- Gauge v2.3 software, Fujifilm). The membrane was stripped Immuno-Sorbent Assay (Invitrogen, #KHO1051). in stripping solution (100 mM Tris-HCl, pH 7.5, 0.9% NaCl, Caspase 3 assay. Caspase 3 activity in cells induced to apopto- 7 μl/ml β-mercaptoethanol, 2% SDS) and then blocked with sis was measured by CaspACE Assay System (Promega, # G7220). 5% nonfat dry milk in TTBS and probed with anti-mouse glyc- Cells were collected at 0 h, 12 h, 24 h, 48 h post serum deprivaton eraldehydes-3-phosphate dehydrogenase antibody (GAPDH, and lysed with RIPA lysis buffer. Each well of a 96 well plate Santa Cruz Biotechnology, #SC-32233) or anti-mouse β-actin contained 32 μl of caspase assay buffer, 2 μl of DMSO, 10 μl of antibody (AbCA M, #ab20272) as a loading control. Protein lev- 100 mM DTT, 20 μl of cell lysates (30 μg), 34 μl of distilled els were visualized as described above. water and 2 μl of the 10 mM DEVD-aminoluciferin labeled with Quantitative PCR. To quanify transcript abundance in chromophore p-nitroaniline substrate (DEVD-pNA). After 4 h N2amiRdual cells, genes reported to interact and/or associate incubation at 37°C, the release of pNA from the substrate by C 30-33 with PrP were selected and their abundance was determined caspase 3 (DEVDase) was detected using microtiter plate reader by qPCR (Table 1). Total RNA from clonal expanded cells was at 405 nm. extracted using Trizol reagent (Invitrogen, #15596-018) and sin- Apoptosis assay. Cells showing apoptotic change were TM gle stranded cDNA was synthesized using SuperScript III First- detected based on the translocation of membrane phosphatidyl- Strand Synthesis System (Invitrogen, #18080-051) with Oligo dT serine (PS) from the inner space to the surface of the cells. When following the manufacturer’s instructions. ORFs corresponding PS is displayed externally, it can be detected by PS-affinitive to each target gene were amplified by PCR using specific primers Annexin V which has a fluorescent conjugate. The proportion (Table 3) and each PCR product was serially diluted and used to of cells undergoing apoptosis in each cell line under both normal generate standard curves. The primers for qPCR were designed growing condition and following serum deprivation was quan- TM by D-LUX Designer (Invitrogen) (Table 3). Thermal cycling tified using Annexin V-Cy3 apoptosis detection kit (Biovision, 100 Prion Volume 5 Issue 2 Table 3. Primer sequences used in this study Target Gene Purpose Label Sequence (5' to 3') Size Product Size - F TGC TGC TGC CCG ACA ACC ACT ACC TGA GCA 30 miR cassette PCR 412 - R ATC AGC GAA CCG CGG GCC CTC TAG ATC AAC 30 - F GAT CAC TCC TAA AGG AGG AAC AGC TAG AG 29 PCR 643 - R ATA AGG GAG ACG GTC ATG TCA CCA C 25 6720460 F02Rik FAM F CGT TAC TCA GCC TGA ACC TCG GTA A 25 qPCR 68 - R CTG CTC TGG CTC TGG ACT TTC C 22 - F ATG CTG GCA GAT AAC CTA GTG GAG G 25 PCR 1,240 - R AAC TGA TGG ACT CTG GGC TTA CAG G 25 Cdr34 FAM F CGT GGA GAG CTA CTG AAG AAG TGC CA 26 qPCR 67 - R AGG TCT GCA CAG CCT TGT GTG 21 - F AAG CAG GGC CAG AGT TTA CAC AGA T 25 PCR 1,137 - R GCT GGA ACA GGT ATC CCA AGT GAG T 25 Csnk2a1 FAM F CGA AGT TTC TGG ACA AGC TGC TT 23 qPCR 104 - R AGC CTG GTC CTT CAC AAC AGT G 22 - F ACG CTT CTC CTT GTC TCG AAT GAC T 25 PCR 1,178 - R GCT CCT GCT TCG AGT CCT TAA TGA C 25 Gfap FAM F CGG ATA CTT TCT CCA ACC TCC AGA TC 26 qPCR 77 - R CCT CTT GAG GTG GCC TTC TGA C 22 - F GGG CAG AGG ATC CCT AAA ACC GGT G 25 PCR 942 - R CAG GCT GTT TGC TGA GGC TGA TCC A 25 Mpg FAM F CGA CAG CCC TAA AGA GAG ACT CCT GT 26 ©2011 Landes Bioscience. qPCR 74 - R GGT CCT CTG GGC TGG AGA AGT 21 - F CTA TGA AGC CAC TGA CAC CGA GTC T 25 PCR 1,629 D - o Rnot d GAA Gi Cs G AGt G Tr AA Gib TA CAu A GCt A Ce TG C. 25 Plk3 - F CTT GGT GAG TGG CCT CAT GC 20 qPCR 75 FAM R CGG AGT GAA ACT ACA GGA GCC TC 23 - F CTG CTG GCC CTC TTT GTG ACT ATG T 25 PCR 736 - R CGA TCA GGA AGA TGA GGA AGG AGA T 25 Prnp FAM F CGA AGC AGC AAC CAG AAC AAC TT 23 qPCR 65 - R ACC GTG TGC TGC TTG ATG GT 20 - F AGG AGG ACA TTG ATA CCC GCA AAA T 25 PCR 1,273 - R ATG TTT TCT GAA GGA TGC CAA GCT G 25 Ppp2r2b FAM F CGG TCT TCT TCA GGA TGT TCG AC 23 qPCR 191 - R AGG ATG CCA AGC TGT ATG CAA G 22 #K102-100). After adding 5 μl of Annexin V-Cy3 into each cell to be significant if probability values of p < 0.05 or p < 0.01 were suspension, apoptotic cells bound to Annexin V were detected obtained. TM Acknowledgments by flow cytometry (BD FACSCalibur Flow Cytometer, BD Bioscience) at 570 nm emission wavelengths. In parallel, control This study was supported by BK21 for Veterinary Science, cell suspension without Annexin V-Cy3 were also analyzed. KRF-2006-005-J502901 and Research Institute of Veterinary Statistical analysis. All experiments were performed at least Science, Seoul National University, Korea and by the Alberta three times. Statistical analysis was performed by ANOVA and Prion Research Institute for the work performed at the Centre for LSD (SPSS Software version 17.0, SPSS Inc.). Data were pre- Prions and Protein Folding Diseases at the University of Alberta, sented as mean ± standard deviation. Differences were considered Canada. www.landesbioscience.com Prion 101 24. 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Ex vivo propagation of 1997; 276:952-5. infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein. Proc Natl Acad Sci USA 2001; 98:4055-9. 102 Prion Volume 5 Issue 2 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Prion Taylor & Francis

Establishment and characterization of Prnp knockdown neuroblastoma cells using dual microRNA-mediated RNA interference

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Taylor & Francis
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Copyright © 2011 Landes Bioscience
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1933-690X
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1933-6896
DOI
10.4161/pri.5.2.15621
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Abstract

RESEARCH PAPER RESEARCH PAPER Prion 5:2, 93-102; April/May/June 2011; © 2011 Landes Bioscience Establishment and characterization of Prnp knockdown neuroblastoma cells using dual microRNA-mediated RNA interference 1,2,† 1,† 2 2 2 2 3 Sang-Gyun Kang, Yu-Mi Roh, Agnes Lau, David Westaway, Debbie McKenzie, Judd Aiken, Yong-Sun Kim 1, and Han Sang Yoo * Department of Infectious Diseases; College of Veterinary Medicine; KRF Zoonotic Disease Priority Research Institute and BK21 Program for Veterinary Science; 2 3 Seoul National University; Seoul, Korea; Centre for Prions and Protein Folding Diseases; University of Alberta; Edmonton, AB Canada; Ilsong Institute of Life Science and Department of Microbiology; College of Medicine; Hallym University; Anyang, Korea These authors contributed equally to this work. Key words: N2a, miRNA, PrP , Prnp, RNAi Prion diseases are fatal transmissible neurodegenerative disorders. In the pathogenesis of the disease, the cellular prion C Sc Sc protein (PrP ) is required for replication of abnormal prion (PrP ), which results in accumulation of PrP . Although there have been extensive studies using Prnp knockout systems, the normal function of PrP remains ambiguous. Compared with conventional germline knockout technologies and transient naked siRNA-dependent knockdown systems, newly constructed durable chained-miRNA could provide a cell culture model that is closer to the disease status and easier to achieve with no detrimental sequelae. The selective silencing of a target gene by RNA interference (RNAi) is a powerful approach to investigate the unknown function of genes in vitro and in vivo. To reduce PrP expression, a novel dual targeting-microRNA (miRdual) was constructed. The miRdual, which targets N- and C-termini of Prnp simultaneously, more ©2011 Landes Bioscience. effectively suppressed PrP expression compared with conventional single site targeting. Furthermore, to investigate the C C cellular change following PrP depletion, gene expression analysis of PrP interacting and/or associating genes and several assays including proliferation, viability and apoptosis were performed. The transcripts 670460F02Rik and Plk3, Ppp2r2b Do not distribute. and Csnk2a1 increase in abundance and are reported to be involved in cell proliferation and mitochondrial-mediated apoptosis. Dual-targeting RNAi with miRdual against Prnp will be useful for analyzing the physiological function of PrP in neuronal cell lines and may provide a potential therapeutic intervention for prion diseases in the future. C 8,9 physiological role of PrP Introduction . These transgenic animal models are, 10,11 however, expensive and time consuming. To overcome these Prion diseases are group of fatal transmissible neurodegenerative problems, cell culture models of prion diseases have been used for disorders characterized by a long incubation period and broad screening and elucidating the mechanism of action of anti-prion neuropathology including spongiform encephalopathy, glio- agents and to analyze biological properties of PrP at the molecu- 1 7,11,12 sis and neuronal loss. They are classified into sporadic, inher - lar and cellular levels. Elucidating the cellular function of ited and acquired forms, which can spread within and between PrP may help in deciphering mechanisms of prion pathogenesis 2 5,13 mammalian species. The central feature of prion diseases is the and in devising therapeutic strategies. C C conversion of a host-encoded prion protein (PrP ) into a dis- Mature PrP translocates to the outer leaflet of the plasma Sc ease-associated isoform (PrP ), which shares same amino acid membrane in proximity to raft-associated signaling molecules. sequence with PrP but is posttranslationally converted to the It traffics in and out of lipid rafts and is involved in a diversity 14-16 infectious form, accumulating primarily in the central nerve sys- of molecular and physiological functions. These proper- 3 C C tem. PrP is implicated in prion pathogenesis as it is required for ties of PrP suggest the possibility that several changes in Prnp propagation and development of prion pathology. Although loss knockout cell culture models may be caused by an altered expres- C Sc of PrP function or deposition of PrP is not sufficient to cause sion of interlinked genes with different cellular functions. Prnp Sc the disease, the process of conversion into PrP may alter signal knockouts could reflect not only the missing function but also 5-7 transduction, thereby giving a toxic dominant function. the compensation for that missing function, as gene deletions in There have been extensive efforts to develop prion disease individual cells or tissues are often accompanied by compensa- 17,18 models in vivo, especially Prnp knockout models for defining the tory changes in gene expression patterns. *Correspondence to: Han Sang Yoo; Email: [email protected] Submitted: 09/20/10; Accepted: 03/28/11 DOI: 10.4161/pri.5.2.15621 www.landesbioscience.com Prion 93 Results Establishment of stable Prnp knockdown cells. Knockdown of a gene using exogenously introduced siRNA is always transient due to a cell division and/or degradation of siR NA molecules. To develop stable Prnp knockdown cells, small hairpin struc- tural artificial miRs were designed ( Fig. 1A). The direction and sequence of the artificial miRs were confirmed by sequence analysis, and N2a cells were transfected with the either miR1, miR2, miRdual or miRscr constructs. The transient effects of each miR on prion protein expression were assessed by western blot analysis. The miRdual most effectively knocked down the expression of PrP by 75 ± 2% compared with wild-type N2a cells (p < 0.01), whereas PrP level in N2a cells transfected with miR1, miR2 and miRscr was decreased by 17 ± 7%, 24 ± 9% (p < 0.05) and no knockdown effect, respectively (Fig. 1B and C). To establish stable Prnp knockdown cells, the N2a cells transfected with miRdual or miRscr were selected with Blasticidin S for a month and are referred to here as N2amiRdua l and N2amiRscr, respectively. The single clones were further selected by limiting dilution and clonally expanded cells were analyzed by genomic PCR, qPCR and western blot to confirm stable transformation with miR as well as transcription and expression of Prnp. Association of the miR plasmid with host chromosomal sequences was assessed by PCR of genomic DNA. ©2011 Landes N2 amB iRdi uao l tes sted pc oi site ive fn or tc he 5e 50 b. p amplicon, while Figure 1. Schematic features of miR cassettes and knockdown efficacy N2amiRscr yielded 412 bp amplicon. No amplicon occurred in N2a cells. (A) Each miR cassette in pcDNA6.2-GW/EmGFP vector. with genomic DNA from wild-type N2a cells (Fig. 2A). To (B) Western blot of N2a cells transfected with each miR expression construct. (C) Quantitation of PrP expression. When miRdual was intro- quantify the effect of miR on Prnp transcription, qPCR was Do not distribute. duced into the cells, PrP expression was the most efficiently decreased used and transcription input of each sample was normalized as compared with wild-type cells (*p < 0.05, **P <0.01). using qPCR of Gapdh. The Prnp transcript was decreased by 87 ± 1% in N2amiRdual (p < 0.01), while it was 50 ± 15% RNA interference (RNAi) refers to the use of 21–23 nucleo- lower in N2amiRscr compared with wild-type N2a (p < 0.05) tide short interfering R NA s (siR NA s) mediating post-transcrip- (Fig. 2B). PrP expression was quantified by western blot. Band tional degradation or translational repression of homologous intensity analysis of western blot indicated that the resultant gene transcripts. Stable knockdowns can be obtained by consti- protein expression of PrP was decreased in N2amiRdual by 96 19,20 tutive expression of the siRNA from the host chromosome. ± 1% and 94 ± 2% in two different cell clones compared with RNAi has been accepted as the most powerful reverse-genetics wild-type (p < 0.01). N2amiRscr showed a reduced expression approach in mammalian cells, however, there are significant of PrP by 27 ± 4% (p < 0.05) (Fig. 2C). technical limitations including identifying efficient methods to Differential gene expression in stably transformed design and deliver siRNA. Unlike other approaches, such as N2amiRdual cells. We next analyzed gene expression changes traditional gene targeting by homologous recombination, anti- in response to Prnp knockdown. These studies were undertaken sense vectors and catalytic RNA or DNA molecules, RNAi is in N2a cells and N2amiRdual derivative and corresponded an endogenous natural pathway and allows cross-species appli- to the quantified expression of several target genes thought to 12 C cation. RNAi, which acutely decreases target expression, also interact and/or associate with PrP (Table 1). For all targets, has the advantage of avoiding compensatory or mechanistic transcript inputs were normalized using Gapdh and standard adaptations. curves were generated using serially diluted PCR products of The present study describes the development of a prion each specific gene. Relative expression levels were expressed as knockdown cell culture model by introducing artificial percent compared to wild-type N2a cells. Of the seven targets microR NA (miR) targeting Prnp into the mouse neuroblastoma selected, 670460F02Rik, Csnk2a1, Plk3 and Ppp2r2b increased cell line (N2a) as well as RK13 cells expressing mouse PrP . in abundance (220 ± 33%, 230 ± 52%, 183 ± 10% and The established Prnp knock-down N2a cells were characterized 519 ± 5%, respectively) in N2amiRdual cells compared with by investigating the expression profiles of the genes known as wild-type N2a cells (p < 0.01) (Fig. 3). The abundance of these PrP interacting and/or associating molecules, proliferation, transcripts also increased, but to a less degree, in N2amiRscr viability and apoptotic resistance. (121 ± 6%, 132 ± 11%, 147 ± 16% and 350 ± 21% respectively). 94 Prion Volume 5 Issue 2 The trend in expression patterns for all target genes was negatively correlated with the amount of PrP in N2amiRdual and N2amiRscr cells (Fig. 2B and C). For Mpg, the mRNA expression was increased both in N2amiRdual and N2amiRscr by 195 ± 21% and 184 ± 24%, respectively (p < 0.05) (Fig. 3). As expected, the Cdr34 and Gfap transcripts as controls were not detectable until 40 cycles of qPCR reac- tion in both N2amiRs and wild-type N2a cells. Alteration in cellular characteristics following PrP k nock-down. A fter estab- lishment of the prion knockdown cell line, N2amiRdual, proliferation, viability and mitochondrial-mediated apoptosis were examined. In proliferation assays, Figure 2. Transfection of miR cassettes and Prnp knockdown efficacy at mRNA and protein level. the N2amiRdual showed decreased pro- (A) PCR of genomic DNA shows miR cassette-specific amplicons. Lane L, 100 bp DNA ladder; lane 1, expression vector with miRdual cassette; lane 2, expression vector with miRscr; lane 3, wild- liferation, to 88 ± 4%, in mitochondrial type cells; lane 4, N2amiRdual; lane 5, N2amiRscr; lane 6, no template. (B) qPCR analysis of PrP dehydrogenase activity tests compared transcript abundance showing knockdown efficiency of miRdual in mRNA levels. (C) Western blot with wild-type (p < 0.05). No significant analysis of PrP protein abundance. Top part is the western blot and bottom panel is densitomet- difference was observed in lactate dehy- ric evaluation. dual1 and dual2 are N2amiRdual cell clones (*p < 0.05, **p < 0.01). drogenase tests for cell viability (Fig. 4A). Based on the observed morphology of the cell body an© d neuri2 tis, n0 o d1 iffe1 renc eL s wea re on bserd ved ie n s (Fi g.B 5C). Tio he ps ropoc rtioi n oe f apn optoc tic ce ells b. ound to Annexin V N2amiRdual and N2amiRscr compared with wild-type. Both was increased by 212 ± 4% in N2amiRdual at 24 h post apoptosis cell types expressed the GFP reporter molecules, however, the induction when compared to the percentage of dying cells before signal was stronger in miRscr cassette than in miRdual (Fig. apoptosis induction (p < 0.05). Most N2amiRscr and wild-type Do not distribute. 4B). GFP signals derived from miR expression cassette were N2a cells were resistant to apoptotic stimulation regardless of attenuated in N2amiRdual cells, likely due to increased pro- growth environment (Fig. 6). cessing of the miRdual. Lentiviral transduction of RK13 expressing mouse PrP To measure mitochondrial-mediated apoptosis induced by with miRs. To confirm the reduction of PrP levels by the dual serum withdrawal, the expression level of mitochondria fission knockdown method, a stable clone of rabbit kidney epithelial 23 C related protein, dynamin related protein 1 (Drp1), was quanti- cells (RK13), expressing both mouse PrP (RK13moPrnp) and fied. Drp1 expression was increased in N2amiRdual by 112 ± GFP was transduced with lentivirus containing either the miR- 4% 24 h following apoptosis induction when compared to the dual or miRscr expression cassettes. The cells were harvested at level before apoptosis induction (p < 0.05). There was, however, 70% and 100% conu fl ence and the relative abundance of PrP no significant difference in the Drp1 expression level between analyzed by western blot (Fig. 7A). In these analyses quanti- N2amiRscr and N2a wild-type, regardless of growth condition ties of PrP and GFP were normalized to the sample with either such as serum deprivation (Fig. 5A). The level of pro-apop- the miRdual or miRscr at 70% cell conu fl ency. PrP level was totic Cytochrome C (Cyt c) in cytosol increased 215 ± 2% in decreased in RK13moPrnp cells with miRdual (46 ± 8% and N2amiRdual cells during serum deprivation compared to the 58 ± 5%), while no significant difference in PrP level was level before serum deprivation (p < 0.05). Again there was no observed in RK13moPrnp cells with miRscr (p < 0.05) (Fig. 7B). difference between N2amiRscr and wild-type N2a cells even Besides assessing protein loading with an actin probe, the GFP though cells had undergone apoptotic stimulation (Fig. 5B). also encoded within the bigenic plasmid was evaluated by use of During apoptotic stimulation, morphological changes between an anti-GFP antibody. No diminution was seen when using the each cell lines were observed by light microscopy. Neurite retrac- “dual” lentivirus, speaking to specificity, and in fact GFP protein tion was induced and proliferation inhibited in N2amiRdual levels were elevated by ~30% both in RK13moPrnp-miRdual cell 24 h post serum deprivation, while N2amiRscr and wild- (136 ± 4% and 139 ± 7%) and in RK13moPrnp-miRscr cells type N2a cells maintained their structural integrity. The caspase (127 ± 6% and 129 ± 4%). This effect cannot represent alle- 3 activity in N2amiRdual cells was increased from 1.6 pmol viation of a transcription interference effect from Prnp cassette pNA liberated/hour at 0 h to 18.8 pmol pNA liberated/hour at within the bigenic pBUD vector (which has tandem expression 48 h post serum deprivation (p < 0.01), whereas there were no cassettes), as it is seen with viruses with different effects upon significant differences in N2amiRscr and wild-type N2a cells PrP expression. www.landesbioscience.com Prion 95 Table 1. Target genes of qPCR for characterizing the established N2amiRdual cell line Gene Gene Name GenBank Accession No. General Putative Function Ref. Symbol C C The function of PrP is not known. PrP is encoded in the host Prnp prion protein NM_011170 genome and is expressed both in normal and infected cells. RIKEN cDNA 6720460F02 gene AK049710 Unknown 30 F02Rik In fission yeast, acts as a mitotic inducer. In G it negatively regu- Cerebellar degeneration- Cdr34 NM_001166658 lates wee1, a mitotic inhibitor. Also has a role in cytokinesis where it 31 related antigen 1 required for proper septum formation. Casein kinases are operationally defined by their preferential utiliza- casein kinase 2, alpha1 Csnk2a1 AK011501 tion of acidic proteins such as caseins as substrates. The alpha and 32 polypeptide alpha’ chains contain the catalytic site. GFAP, a class-III intermediate filament, is a cell-specific marker that, Gfap Glial fibrillary acidic protein AF332061 during the development of the central nervous system, distinguishes 33 astrocytes from other glial cells. Hydrolysis of the deoxyribose N-glycosidic bond to excise 3-meth- N-methylpurine-DNA Mpg NM_010822 yladenine, and 7-methylguanine from the damaged DNA polymer 30 glycosylase formed by alkylation lesions. Serine/threonine protein kinase involved in regulating M phase Plk3 Polo-like kinase 3 (Drosophila) NM_013807 functions during the cell cycle. May also be part of the signaling net- 30 work controlling cellular adhesion. pro© tein pho2 spha0 tase 2 1 1 Landes The B r B egulai toro y subs unit mc igi ht me odn ulate sc ube strate s. electivity and Ppp2r2b (formerly 2A), regulatory sub- NM_028392 catalytic activity, and also might direct the localization of the cata- 31 unit B (PR 52), beta isoform lytic enzyme to a particular subcellular compartment. Do not distribute. Figure 3. Quantitative PCR analysis of PrP interacting and/or associ- ated molecules. 6720460F02Rik, Plk3, Ppp2r2b, Csnk2a1 transcripts were upregulated in prion knockdown N2amiRdual cells compared to wild- type N2a cells. The increased Mpg transcripts in both N2amiRdual and N2amiRscr seemed to have no correlation with decreased PrP (*p < 0.05, **p < 0.01). Discussion The knockdown of PrP in N2a cells using a miR-based RNAi system provides a novel cell culture model for studying prion biology. The artificial miRs were designed to target nucleo - tide residues 36–56 and 668–688 of mouse PrP ORF region, respectively. The miRs were constructed to be expressed as a cluster in a long primary transcript driven by RNA polymerase II. This dual targeting strategy enhanced knockdown efficacy more than 3-fold compared to single site targeting method. It was reported that a single miR can bind to and regulates many different mRNA target, conversely, several different miRs can 25,26 pair with and cooperatively control a single mRNA target. Downregulation of Prnp was also observed in N2a transfected with miRscr, an unexpected result as the scrambled sequence does not target any known vertebrate gene. To better define this targeting effect of miRscr to Prnp, we examined RK13moPrnp cells, which were lentiviral-transduced with miRdual or miRscr, respectively. In the viral-tranduced cells with miRdual, the expression levels of PrP varied inversely with the amount of 96 Prion Volume 5 Issue 2 GFP. These results suggest that, rather than specific targeting by miRscr, the reduced PrP expression in N2amiRscr is likely due to exogenous factors through gene transfection, such as genomic 27-29 insertion of foreign genes that may disrupt cellular processes. An exploration of cellular interactors is one of the way to disclose unknown function of protein of interest. Transcript abundances of PrP interacting and/or associating proteins fol- lowing stable Prnp knockdown can provide valuable information concerned with normal cellular function of PrP . Therefore, we selected seven target genes from previously reported microarray 30-33 and protein microarray data and transcript abundance was investigated by qPCR. Of the seven genes examined in this study, the abundance of four PrP interactors increased following Prnp downregulation. One of the upregulated genes in N2amiRdual is 6720460F02Rik gene, also known as CATS (computer-annotated as FAM64A), the Homo sapiens family with sequence similarity 64, member A in GenBank (NM_144526). Its transcript is abun- dantly expressed in proliferative state brain tissues, glioma and Figure 4. Cell proliferation, viability and GFP expression test. (A) Viability primitive neuroectodermal tumors, however, the precise cellular and proliferation of N2a cells stably transformed with miRdual or miRscr function of the protein is unknown. FAM64A was shown to (*p < 0.05). (B) Confocal microscopy images of clonal expanded cells. interact with PrP through protein microarray and coimmuno- N2amiRdual and N2amiRscr showed green fluorescent signals. Cell nuclei were stained with DAPI (blue). Photos were taken at 20 doubling passages. precipitation with recombinant human PrP spanning amino acid GFP signals stably expressed over 135 doubling passages. Scale bar, 50 um. residues 23–231. In addition, FAM64A is located predominantly in the nucleus, where it colocalizes with the recombinant human C 30 PrP . This suggests that FAM64A/CATS/6720460F02Rik might have a role in c© ontro2 l of c0 ell p1 rol1 ifer atL ion ca onsn istend t wie th s Bioscience. the decreased cell proliferation observed in our studies. The increased transcription level could be a compensatory reaction to the loss of PrP function associated with cell proliferation. Do not distribute. Plk3 is a member of the polo family of serine/threonine kinases and is also as a PrP interacting partner. It localizes to the nucleolus and is involved in regulation of the G /S phase tran- 35,36 C sition. The control of cell proliferation by PrP may alter the expression of cell cycle-related genes, such as cyclin D1, Esp8 and CD44, in a cell type-specific way. The increased level of Plk3 in N2amiRdual cells may be a compensatory response to a PrP - dependent cell proliferation event. Moreover, the overexpression of Plk3 may mediate inhibition of proliferation as a result of the C 36,37 loss of the anti-apoptotic function of PrP . Further analysis into the mechanism of PrP function associated with Plk3 will be required. Ppp2r2b encodes the neuron-specific regulatory beta subunit of the protein phosphatase 2A (PP2A) holoenzyme and the muta- tions in Ppp2r2b are responsible for the neurodegenerative disor- der, spinocerebellar ataxia 12. Increased expression of Ppp2r2b reported to induce mitochondrial fragmentation through stimu- lating mitochondrial fission protein such as Drp1, which can lead 38,39 to neuronal apoptosis. Our observation of increased expres- sion of Ppp2r2b following PrP knockdown was not consistent Figure 5. Apoptotic resistance to serum deprivation. Cells were cultured under conditions of serum deprivation and collected at indicated time points. (A) Detection of Drp1 expression levels analyzed by densitometric analysis of western blot. (B) Cytochrome C expression in cytosolic frac- tions detected by immunoassay. (C) Analysis of caspase 3 activity calcu- lated by comparison with the free pNA. dual, N2amiRdual; scr, N2amiRscr; wt, miR non-treated wild-type N2a (*p < 0.05, **p < 0.01). www.landesbioscience.com Prion 97 ©2011 Landes Bioscience. Do not distribute. Figure 6. Detection of apoptotic cells following serum deprivation using Annexin V-Cy3 assay. Cells were collected at the indicated time points and 5 x 10 cell suspensions were treated with Annexin V. The proportion of cells showing apoptotic change was increased in N2amiRdual whereas most of cells showed the resistance against apoptosis stimulation in N2amiRscr and wild-type N2a cells (*p < 0.05). C 43,44 with previous study that showed the increased Ppp2r2b in a PrP by environmental genotoxins. The upregulation of Mpg both overexpressing HEK293 cell line. It is conceivable that dysreg- in N2amiRdual and N2amiRscr is curious, but may be a response ulation of Ppp2r2b via abnormal expression of PrP might be a to the expression of double-stranded miR that has internal loops. critical cause of cell death secondary to mitochondrial dysfunction. There was no difference observed in viability of the N2a lines Mitochondrial fragmentation by overexpression of Ppp2r2b occurs but proliferation was decreased in N2amiRdual compared with upstream of apoptosis and was sufc fi ient for hippocampal neuron N2amiRscr and wild-type cells. This is likely due to the down- 39 C death. It is consistent that transfection of PrP knockout cells with regulation of PrP rather than other exogenous factors. It has 37 C a Prnp expression vector prevented typical apoptotic changes. been shown that the expression level of PrP was positively cor- 37,45 Casein kinase 2 (CK2) participates in the regulation of diverse related with the cell proliferation. In agreement with other 46-48 cellular processes. It is a messenger-independent protein serine/ studies of prion knockout model, newly constructed prion threonine kinase and especially abundant in the brain. It is local- knockdown N2amiRdual cells were more vulnerable to apop- C 40 ized to the outer plasma membrane where PrP also resides. The totic stimulation than N2amiRscr and wild-type N2a. Inhibition catalytic α subunit of CK2 (CK2α), encoded by the Csnk2a1 gene, of Drp1 activity during apoptosis in mammalian cells inhibits C 41 is reported to bind to cytosolic non-glycosylated PrP . CK2α mitochondrial fragmentation, Cyt c release and the rate of cell 39,49,50 also binds to PP2A in mitogen-starved cells in vitro and overex- death. Altering the mitochondrial fusion/fission balance pression of CK2α results in suppression of cell growth. These towards fission by Drp1 could lead to cell death by promoting reports are consistent with the result of decreased proliferation mitochondrial release of pro-apoptotic proteins including Cyt c. in N2amiRdual cells that may cause compensatory increases of The release of Cyt c from mitochondrial inter-membrane space C 51,52 Ppp2r2b and Csnk2a1 following decreased expression level of PrP . to the cytosol has reported to activate the caspase family, an The Mpg gene encodes the base-excision repair enzyme, important event for the downstream activation of apoptosis cas- 49,53 3-methyladenine DNA glycosylase and has been reported as a cade. This phenomenon is supported by Cyt c knockout cell PrP interacting protein. Mpg plays a vital role in preserving cer- lines study showing reduced caspase 3 activation and resistance ebellar development and protecting mature neurons from insults to various apoptotic stimuli. 98 Prion Volume 5 Issue 2 In conclusion, the stable N2amiRdual cells will be a useful tool for elucidating the physiological function of PrP and neu- ropathogenesis of prion disease. Moreover, since miR is known as more suitable approach for achieving RNAi in mouse brain, in terms of toxicity, it is expected that the dual targeting artifi - cial miR-based RNAi used in this study will provide a promising therapeutic intervention for prion diseases and other neurodegen- erative disorders. Materials and Methods Cell culture. N2a and RK13moPrnp cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) with high glu - cose (4.5 g/l) and 2 mM glutamine (Gibco , Invitrogen, #11995), supplemented with 10% fetal bovine serum, penicillin and strep- tomycin. Cells were cultured at 37°C in a 5% CO incubator. Designing artificial miRNAs. miRs were designed to knock- down the endogenous Mus musculus Prnp in N2a cells based on previous report in reference 56. The miR design algorithm, provided by the Invitrogen web-site, was used to select target sequences. The basic local alignment search tool (BLAST) was used with the mouse genome database to identify unique regions in the murine Prnp open reading frame (ORF, NM_011170). Two regions were chosen to target; the N-(miR1) and C-termini (miR2) of the Prnp ORF. miR1 and miR2 bind at 191 to 211 and 823 to 843 of Pr© np O2 RF re0 gio1 n, re1 sp ecL tivea ly. En ach sid ngle e-s Bioscience. stranded DNA (ssDNA) was designed to contain the antisense C Figure 7. Downregulation pattern of PrP in RK13-moPrnp cells sequence derived from target sites, followed by 19 nucleotides to harvested at different confluences. RK13-mo Prnp cells were lentiviral- transduced with miR expression cassettes and the relative abundances form the terminal loop and sense target sequence with 2 nucleo- Do not distribute. of PrP and GFP processed were determined by western blot (*p < 0.05, tides removed to create an internal loop (Table 2). **p < 0.01). Construction and expression of miRNAs. The miRs were synthesized commercially (Invitrogen) and annealed for direc- tional cloning. The synthesized top- and bottom-strand DNAs and analyzed by expression of green fluorescent protein (GFP) (Table 2) were annealed and ligated into pcDNA6.2-GW/ reporter gene, genomic PCR, quantitative PCR (qPCR) and EmGFP-miR (Invitrogen, #K4936-00) expression vector. The western blot. ligation mixture was transformed into TOP10 competent E. coli. Transfection of RK13 cells with mouse Prnp. RK13 cells To confirm the orientation and DNA sequence of the miRs, each were transfected with a derivative of the bigenic pBud mamma- construct was sequenced using an automated DNA Sequencer lian vector expressing both GFP and a wt mouse Prnp coding (ABI PRISM 377 L). To express miRs as one primary transcript, region. This procedure used lipofectamine2000 (Invitrogen, miR2 cassette were digested and ligated into miR1 construct. #11668). Cells stably expressing mouse PrP (RK13moPrnp) The linked miRs (miRdual) was transformed and analyzed as were selected with Zeocin (Invitrogen, #R250) and screened by described above. A miR cassette containing a scramble sequence western blot. (miRscr) (Invitrogen, #K4936-00), which can be processed into Production of lentiv ir us a nd infection of R K13moPrnp. The mature miR but is not predicted to target any known vertebrate m i R e x pre s sion c a s set te s i nc lud i ng GFP were c loned i nto pL ent i6 / gene, was used as a negative control (Fig. 1A). V5-DEST lentiviral vectors (Invitrogen, #K4937) by Gateway Transfection of N2a cells with miRNAs. The wild-type N2a site-specific recombination. The constructed lentiviral vectors cell line was transfected with the expression construct using the (3 μg) were co-transfected with a third-generation lentivirus FuGENE6 transfection reagent (Roche, #11814443001). At 24 h packaging vector (9 μg, Invitrogen, # K4970) into HEK293FT post transfection, the cells were lysed with RIPA lysis buffer cells using Lipofectamine 2000 reagent (Invitrogen, #11668). (1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, The viral supernatant was collected 48 h after co-transfection 50 mM Tris-HCl, pH 7.4, 0.1% SDS, 1 mM EDTA) contain- and concentrated by ultracentrifugation for 2 h at 153,725 g and ing a protease inhibitor cocktail (Amresco, #M222) and the 4°C. The titer of lentiviruses was determined using HT1080 efficiency of downregulation of Prnp was measured by western cells and RK13moPrnp cells were infected with the virus par- blot. Through drug selection with Blasticidin S (Invitrogen, ticles at an MOI of 10. After Blasticidin S selection for two #R 21001) and limiting dilution, clonal expanded cells expressing weeks, cells were analyzed for knockdown of PrP by western miRdual (N2amiRdual) or miRscr (N2amiRscr) were obtained blot. www.landesbioscience.com Prion 99 Table 2. Sequences of artificial miRs targeting Mus musculus Prnp ORF Sequence L Mature miRNA Sequence Loop Sequence Sense Sequence L T TGC TG ACA TCA GTC CAC ATA GTC ACA GTT TTG GCC ACT GAC TGA C TGT GAC TAT GGA CTG ATG T miR1 B C TGT AGT CAG GTG TAT CAG TGT CAA AAC CGG TGA CTG ACT G ACA CTG ATA CCT GAC TAC A GTC C T TGC TG ATC TTC TCC CGT CGT AAT AGG GTT TTG GCC ACT GAC TGA C CCT ATT ACC GGG AGA AGA T miR2 B C TAG AAG AGG GCA GCA TTA TCC CAA AAC CGG TGA CTG ACT G GGA TAA TGG CCC TCT TCT A GTC C T TGC TG AAA TGT ACT GCG CGT GGA GAC GTT TTG GCC ACT GAC TGA C GTC TCC ACG CAG TAC ATT T miRscr B C TTT ACA TGA CGC GCA CCT CTG CAA AAC CGG TGA CTG ACT G CAG AGG TGC GTC ATG TAA A GTC C L, linker; T, top strand; B, bottom strand. Genomic PCR. Genomic DNA of clonal expanded cells was iso- for the ABI PRISM 7300 (Applied Biosystems) was performed lated using a genomic DNA puric fi ation kit (Promega, #A11120). as follows: 2 min at 50°C for uracil N-glycosylase (UDG) incu- Primer pairs were designed to amplify specic fi miR cassettes bation which removes uracil-containing products from previous (Table 3). The PCR parameters consisted of an initial denatur- reactions, UDG inactivation and DNA polymerase activation for ation step at 95°C for 5 min followed by 40 cycles of denaturation 2 min at 95°C, followed by 40 cycles of 15 sec at 95°C for dena- at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at turation and 30 sec at 60°C for hybridization and elongation. 72°C for 1 min and a final extension at 72°C for 15 min. PCR Proliferation and viability assay. The established N2a- products were analyzed by electrophoresis on 1.0% agarose gel. miRdual, N2amiRscr and wild-type cell lines were cultured in Western blot. For western blot analysis, 5 μg of pro- 96-well plates with 3 x 10 cells in 100 μl of DMEM culture tein from each cell lysate was prepared and fractionated on a medium per each triplicate well. Activity of cellular mitochon- 12% Tris-glycine gel and transferred to a 0.2 micron nitro- drial and lactate dehydrogenase were determined using commer- cellulose membrane using a dry blotting system (Invitrogen, cial kits (Biovision, #302-500, #313-500) to measure proliferation #IB1001). The mem© brane w2 a0 s blo1 cke1 d w iL th 5a % nn onfd at de ry s and c B ells vi iao bilits y rec speci tie vely. An ftec r 4e 8 h i. ncubation, the tetra- milk in TritonX-Tris buffered saline (TTBS: 0.1% TritonX-100 zolium salt (WST) was added to each well and the amount of in 100 mM Tris-HCl, pH 7.5, 0.9% NaCl) and then probed formazan production quantified using a microtiter plate reader at with anti-PrP antibody, 3F10, anti-GFP antibody (AbCAM, 450 nm. Non-viable controls were prepared by treating cells with Do not distribute. #ab290) or anti-dynamin-related protein 1 (Drp1) antibody 1% TritonX-100. (BD Transduction Laboratories, #611112) at 4°C for over- Cytochrome C assay. The cytosolic and mitochondrial frac- night. An anti-mouse IgG conjugated to horseradish peroxi- tions from each cell line were separated using a mitochondria iso- dase (Santa Cruz Biotechnology, #SC-2005) was used as the lation kit (Pierce, #89874) following manufacturer’s instruction. secondary antibody. The bound antibodies were visualized by Total protein concentration of the cy tosolic fractions were adjusted chemiluminescence (Amersham, #RPN2106) and protein lev- using BCA protein assay (Pierce, #23227) and the amount of Cyt els were measured by scanning and evaluating density (Multi c in each fraction were analyzed by sandwich Enzyme Linked- Gauge v2.3 software, Fujifilm). The membrane was stripped Immuno-Sorbent Assay (Invitrogen, #KHO1051). in stripping solution (100 mM Tris-HCl, pH 7.5, 0.9% NaCl, Caspase 3 assay. Caspase 3 activity in cells induced to apopto- 7 μl/ml β-mercaptoethanol, 2% SDS) and then blocked with sis was measured by CaspACE Assay System (Promega, # G7220). 5% nonfat dry milk in TTBS and probed with anti-mouse glyc- Cells were collected at 0 h, 12 h, 24 h, 48 h post serum deprivaton eraldehydes-3-phosphate dehydrogenase antibody (GAPDH, and lysed with RIPA lysis buffer. Each well of a 96 well plate Santa Cruz Biotechnology, #SC-32233) or anti-mouse β-actin contained 32 μl of caspase assay buffer, 2 μl of DMSO, 10 μl of antibody (AbCA M, #ab20272) as a loading control. Protein lev- 100 mM DTT, 20 μl of cell lysates (30 μg), 34 μl of distilled els were visualized as described above. water and 2 μl of the 10 mM DEVD-aminoluciferin labeled with Quantitative PCR. To quanify transcript abundance in chromophore p-nitroaniline substrate (DEVD-pNA). After 4 h N2amiRdual cells, genes reported to interact and/or associate incubation at 37°C, the release of pNA from the substrate by C 30-33 with PrP were selected and their abundance was determined caspase 3 (DEVDase) was detected using microtiter plate reader by qPCR (Table 1). Total RNA from clonal expanded cells was at 405 nm. extracted using Trizol reagent (Invitrogen, #15596-018) and sin- Apoptosis assay. Cells showing apoptotic change were TM gle stranded cDNA was synthesized using SuperScript III First- detected based on the translocation of membrane phosphatidyl- Strand Synthesis System (Invitrogen, #18080-051) with Oligo dT serine (PS) from the inner space to the surface of the cells. When following the manufacturer’s instructions. ORFs corresponding PS is displayed externally, it can be detected by PS-affinitive to each target gene were amplified by PCR using specific primers Annexin V which has a fluorescent conjugate. The proportion (Table 3) and each PCR product was serially diluted and used to of cells undergoing apoptosis in each cell line under both normal generate standard curves. The primers for qPCR were designed growing condition and following serum deprivation was quan- TM by D-LUX Designer (Invitrogen) (Table 3). Thermal cycling tified using Annexin V-Cy3 apoptosis detection kit (Biovision, 100 Prion Volume 5 Issue 2 Table 3. Primer sequences used in this study Target Gene Purpose Label Sequence (5' to 3') Size Product Size - F TGC TGC TGC CCG ACA ACC ACT ACC TGA GCA 30 miR cassette PCR 412 - R ATC AGC GAA CCG CGG GCC CTC TAG ATC AAC 30 - F GAT CAC TCC TAA AGG AGG AAC AGC TAG AG 29 PCR 643 - R ATA AGG GAG ACG GTC ATG TCA CCA C 25 6720460 F02Rik FAM F CGT TAC TCA GCC TGA ACC TCG GTA A 25 qPCR 68 - R CTG CTC TGG CTC TGG ACT TTC C 22 - F ATG CTG GCA GAT AAC CTA GTG GAG G 25 PCR 1,240 - R AAC TGA TGG ACT CTG GGC TTA CAG G 25 Cdr34 FAM F CGT GGA GAG CTA CTG AAG AAG TGC CA 26 qPCR 67 - R AGG TCT GCA CAG CCT TGT GTG 21 - F AAG CAG GGC CAG AGT TTA CAC AGA T 25 PCR 1,137 - R GCT GGA ACA GGT ATC CCA AGT GAG T 25 Csnk2a1 FAM F CGA AGT TTC TGG ACA AGC TGC TT 23 qPCR 104 - R AGC CTG GTC CTT CAC AAC AGT G 22 - F ACG CTT CTC CTT GTC TCG AAT GAC T 25 PCR 1,178 - R GCT CCT GCT TCG AGT CCT TAA TGA C 25 Gfap FAM F CGG ATA CTT TCT CCA ACC TCC AGA TC 26 qPCR 77 - R CCT CTT GAG GTG GCC TTC TGA C 22 - F GGG CAG AGG ATC CCT AAA ACC GGT G 25 PCR 942 - R CAG GCT GTT TGC TGA GGC TGA TCC A 25 Mpg FAM F CGA CAG CCC TAA AGA GAG ACT CCT GT 26 ©2011 Landes Bioscience. qPCR 74 - R GGT CCT CTG GGC TGG AGA AGT 21 - F CTA TGA AGC CAC TGA CAC CGA GTC T 25 PCR 1,629 D - o Rnot d GAA Gi Cs G AGt G Tr AA Gib TA CAu A GCt A Ce TG C. 25 Plk3 - F CTT GGT GAG TGG CCT CAT GC 20 qPCR 75 FAM R CGG AGT GAA ACT ACA GGA GCC TC 23 - F CTG CTG GCC CTC TTT GTG ACT ATG T 25 PCR 736 - R CGA TCA GGA AGA TGA GGA AGG AGA T 25 Prnp FAM F CGA AGC AGC AAC CAG AAC AAC TT 23 qPCR 65 - R ACC GTG TGC TGC TTG ATG GT 20 - F AGG AGG ACA TTG ATA CCC GCA AAA T 25 PCR 1,273 - R ATG TTT TCT GAA GGA TGC CAA GCT G 25 Ppp2r2b FAM F CGG TCT TCT TCA GGA TGT TCG AC 23 qPCR 191 - R AGG ATG CCA AGC TGT ATG CAA G 22 #K102-100). After adding 5 μl of Annexin V-Cy3 into each cell to be significant if probability values of p < 0.05 or p < 0.01 were suspension, apoptotic cells bound to Annexin V were detected obtained. TM Acknowledgments by flow cytometry (BD FACSCalibur Flow Cytometer, BD Bioscience) at 570 nm emission wavelengths. In parallel, control This study was supported by BK21 for Veterinary Science, cell suspension without Annexin V-Cy3 were also analyzed. KRF-2006-005-J502901 and Research Institute of Veterinary Statistical analysis. All experiments were performed at least Science, Seoul National University, Korea and by the Alberta three times. Statistical analysis was performed by ANOVA and Prion Research Institute for the work performed at the Centre for LSD (SPSS Software version 17.0, SPSS Inc.). Data were pre- Prions and Protein Folding Diseases at the University of Alberta, sented as mean ± standard deviation. Differences were considered Canada. www.landesbioscience.com Prion 101 24. 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Prion – Taylor & Francis

Published: Apr 1, 2011

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