Philippine Journal of Science 149 (1): 21-26, March 2020 ISSN 0031 - 7683 Date Received: 30 Sep 2019 Importin-β Proteins Expression Modulation in Embryonic Stem Cells and Embryonic Fibroblasts of Mouse Percival P. Sangel* Institute of Animal Science, College of Agriculture and Food Science University of the Philippines Los Baños, College 4031 Laguna, Philippines Importin-β proteins are transport proteins important in the shuttling of cargo proteins by binding to either nuclear localization signal (NLS) or nuclear export signal (NES). This study investigated the in vitro expression modulation of selected importin-βs. Specifically, this study characterized the culture behavior of mouse embryonic stem cells after knockdown of selected importin-β proteins like Cse1L, IPO7, KPNB1, RanBP16, RanBP17, or XPO4. Also, this study assessed the effects of overexpressing RanBP17 or IPO7 during cellular reprogramming of mouse embryonic fibroblasts (MEFs). Results showed that Cse1L and KPNB1 are essential for the viability of mouse embryonic stem cells since knockdown of either one of these proteins resulted in the death of mouse embryonic stem cells. Meanwhile, the growth characteristics of RanBP17, XPO4, IPO7, or RanBP16 knockdown mouse embryonic stem cells were comparable with the control. Aside from round colonies, the appearance of flat cells and spreading growth characteristics in some colonies were observed, which indicated early signs of differentiation. On the other hand, the number of colonies with overexpressed Oct4, Sox2, Klf4, cMYC (OSKM) + RanBP17, or OSKM+ IPO7 was comparable to OSKM+Flag or OSKM (controls). This suggests that RanBP17 or IPO7 has limited application in the generation of induced pluripotent stem cells. Keywords: cellular reprogramming, importin-β proteins, knockdown INTRODUCTION reports available for importin-α contributing to a better understanding of its roles as a transporter. The previous Many intricate trafficking pathways involve the works of Sekimoto et al. (1997) lead to the identification transport of various substances in a living cell. In the of the different subtypes of importin-α. This opened case of the eukaryotic cell, its biomembrane creates a research platform to identify and analyze respective compartmentalization, giving rise to the different NLS in different cargo proteins and elucidate the specific organelles and affecting the transport of all cargo mechanisms involved in their nuclear transport. The molecules. Central to these transport processes is the study of Yasuhara et al. (2007) showed that functional nucleocytoplasmic transport system. This regulates the switching of the importin-α subtype may lead to neural shuttle of the different cargo proteins in and out of the lineage differentiation of embryonic stem cells. Their nucleus as they pass through the nuclear pore complex study also demonstrated significance in the regulation (NPC) of the nuclear membrane. The major key players of the activity of importin-α subtypes, which influence mediating most cargo protein transport via this system mostly the transport of transcription factors as protein are small proteins collectively called importins (alpha cargoes. Moreover, the same report indicated the role of and beta). Between the two importins, there are more importin-αs as indicators of cell lineage determination. *Corresponding Author: [email protected] 21 Philippine Journal of Science Sangel: Importin-β Proteins Expression Vol. 149 No. 1, March 2020 Modulation in Mouse Embryonic Cells The importin-β family also plays an important role in RNA Extraction nucleocytoplasmic transport system. This family consists of For mouse embryonic stem cells, the total RNA was soluble proteins that facilitate majority of macromolecular extracted using TRIZOL (Invitrogen), after which the nuclear transport. In mice, there are more than 20 proteins extracted RNA was DNase-treated (Zymo Research) identified under this family. Specifically, these importin-βs and then reverse-transcribed using the Transcriptor First are associated with nuclear transport of proteins and RNAs Strand cDNA Synthesis Kit (Roche). All procedures via the NPC. Importin-βs may function either as importins followed the manufacturer’s recommendations. or exportins. This is through recognition of either the NLS or NES, respectively, during the nuclear transport of cargo Reverse Transcription PCR and Quantitative PCR proteins. Although, some of them may have dual functions For cDNA synthesis, reverse transcription was done with (Ström and Weis 2001, Chook and Blobel 2001, Yuh and polymerase chain reaction (PCR) conditions set at 25 °C Suel 2011). This transport mechanism is regulated by for 10 min, 50 °C for 60 min, and 85 °C for 5 min. And Ran (RanGTP), which is a member of Ras superfamily then, qPCR analysis was done using a 384-well plate in (Maherali et al. 2007). ABI PRISM 7900HT system (Applied Biosystems) with Like importin-α, importin-βs are also involved in FastStart™ Universal SYBR Green Master [Rox] (Roche). different cellular events like pluripotency or cell fate The qPCR conditions were set at a holding temperature determination as seen in the studies using embryonic of 95 °C for 30 s, and 40 cycles of 95 °C for 15 s, 60 °C stem cells (Sangel et al. 2014). Thus, importin-βs as for 30 s, and a standard dissociation stage. transporters are biologically important (Yuh and Suel For all the target genes, standard curves were generated. 2011), although there is also report available involving Extracted RNA from mouse embryonic stem cells at 0.8, importin-β protein in non-transport function vitally needed 4, 20, and 100 ng were used for serial dilutions. And then, for cellular viability (Mosammaparast and Pemberton 20 ng from the extracted total of siRNA-treated cells was 2004). This study demonstrated the effects of expression used as template. The Pfaffl method was used to determine modulation of some importin-β proteins in different cell the relative target mRNA expression levels and all values types. Specifically, this study characterized the culture were normalized with respective mRNA levels of GAPDH. behavior of mouse embryonic stem cells after knockdown of selected importin-β proteins. Also, this study assessed the potential use of selected importin-β proteins in cellular Treatment of siRNA Oligonucleotide reprogramming using MEFs. In all of mouse embryonic stem cell transfections, 2 × 105 cells were seeded on the surface of Corning™ polystyrene plates (Sigma) coated with 0.1% gelatin with DMEM supplemented with LIF, 10% FBS, 0.1 mM MATERIALS AND METHODS β-mercaptoethanol (Sigma Chemical), 10 mM of MEM nonessential amino acid (GIBCO), and 100 mM of MEM sodium pyruvate (GIBCO). After cell plating, transfection Mouse Embryonic Stem Cells Culture and was carried out using two different siRNA constructs for Maintenance each target genes (i.e., Cse1L, IPO7, KPNB1, RanBP16, Mouse embryonic stem cells (EB3) (Niwa et al. 1998, RanBP17, and XPO4) with LipofectamineTM RNAiMAX 2002) were used for the knockdown of selected importin-β (Invitrogen). Transfected cells were then incubated at proteins. Mouse embryonic stem cells were grown on 37 °C, in 5% CO2. The medium was changed after 48-h plate surface coated with 0.1% gelatin with Dulbecco’s incubation using 2 mL of the fresh medium not containing modified Eagles as culture medium (DMEM) (Sigma) LIF. This is followed by another transfection following the enriched by addition of leukemia inhibitory factor (LIF), same procedure. Transfected cells were again incubated 10% fetal bovine serum (FBS), 0.1 mM β-mercaptoethanol for another 48 h before they were microscopically (Sigma Chemical), 10 mM of MEM nonessential amino examined, trypsinized, and collected. acid (GIBCO), and 100 mM of MEM sodium pyruvate (GIBCO). The cells were maintained at 37 °C in 5% CO . 2 Plasmids Used in Cellular Reprogramming This study made use of pMXs retroviral vectors expressing MEF Culture and Maintenance transcriptional factors like Oct4, Sox2, Klf4, and c-Myc MEFs were used in cellular reprogramming. MEFs (Addgene) for cellular reprogramming. Importin-βs from C57BL/6NCrSlc (SLC) embryos (12.5–13.5 d – which are highly expressed in mESCs, specifically postcoitum) stored in cryotubes and maintained at –80 °C RanBP17 and IPO7 – were also used. Each of the were thawed and cultured in DMEM supplemented with candidate Importin-β genes was amplified through PCR. 10% FBS. The cells were maintained at 37 °C in 5% CO2. Table 1 shows the respective primer sequences used in 22 Philippine Journal of Science Sangel: Importin-β Proteins Expression Vol. 149 No. 1, March 2020 Modulation in Mouse Embryonic Cells the amplification of target importin-β genes. And then, a re-plated on the surface of mitomycin C-treated feeder cells pMXs-RanBP17 or pMXs-IPO7 construct was made by grown on gelatin-coated 100-mm dish. After another 24 h, inserting the coding sequence of either RanBP17 or IPO7 replacement of culture medium was done using the mouse at BamHI and SalI sites located inside the multi-cloning embryonic stem cells medium containing 20% knockout site of pMXs-Flag plasmid vector (Addgene). After that, serum replacement (Invitrogen). The culture medium was the constructed plasmids were cloned and sequenced. regularly changed every other day until colony formation was visible under microscopic examination. And then, Cellular Reprograming alkaline phosphatase staining was done to count the number The method described by Okada et al. (2010) was used in of colonies of