Development of a Tunable Mammalian Protein Expression

Total Page:16

File Type:pdf, Size:1020Kb

Development of a Tunable Mammalian Protein Expression Development of a Tunable Mammalian Protein Expression System and an Investigation of Promoter Interference in Three Promoters Often Utilized in the Production of Biopharmaceuticals By Nathan R West (MBiomedSci, MSc) Thesis submitted for the degree of Doctor of Philosophy (PhD) Department of Chemical and Biological Engineering University of Sheffield Final submission: August 2014 2 Nathan West The University of Sheffield Thesis Abstract Cell line engineering strategies for improved biopharmaceutical production in mammalian cells often involve the expression of one/multiple genes to try and improve the cellular processes involved in recombinant protein production. Most strategies are relatively simple, involving the use of a strong constitutive promoter for expression of one or more proteins to help increase production. Results often vary and can be cell line and product specific and mean a generic strategy is unlikely to be found. There is a need for more sophisticated expression systems which can express multiple genes but in a controlled fashion and tuned to meet the needs of a specific product. This thesis can be split into two distinct parts but both concern the expression of multiple genes in mammalian cells and recombinant protein production. A tunable mammalian expression system for multi-gene engineering composed of elements of the mammalian unfolded protein response has been developed. ATF6 (activating transcription factor 6) and its binding element (ERSE – ER stress response element) were used to control the expression of the reporter proteins SEAP (secreted alkaline phosphatase) and GFP (green fluorescent protein). By expressing different amounts of ATF6 and by inserting different numbers of ERSEs upstream of a SV40 (Simian virus 40) promoter, driving SEAP/GFP gene transcription, the level of reporter protein expression could be manipulated in a controlled fashion. The system was capable of controlled/tunable expression of both reporter proteins when expressed alone and when they were co-expressed. This a novel use for ATF6 and ERSE and the first step towards the development of a tunable mammalian expression system for multi-gene engineering. This system could also be easily modified to include or use different transcription factors and binding sites as well as having the potential to use completely synthetic components. This work also showed that the presence of ‘promoter interference’ (the negative influence of one promoter on another) could be used to our advantage to increase the range of expression. The SV40 early, human CMV (cytomegalovirus) major immediate-early and human EF1α (elongation factor 1 alpha) are constitutive promoters frequently used in recombinant protein production. The former being used mainly for expression of selection genes and the latter two for strong expression of recombinant proteins. The differences in the strengths of the promoters was demonstrated in CHO (Chinese hamster ovary) cells (CMV > EF1α > SV40) and also their abilities to negatively affect the expression from a co-expressed promoter. The negative influence of one promoter on another is termed ‘promoter interference’. The CMV promoter was shown to have the greatest negative effect on expression from another promoter, decreasing both SEAP mRNA and protein expression, while the SV40 had the least. SEAP expression from the SV40 was reduced the most by the presence of a competing promoter. The level of interference inflicted by a competing promoter (CMV > EF1α > 3 Nathan West The University of Sheffield SV40) seemed to be relative to its strength. This is the first time these three important promoters have been compared in a way which not only demonstrates their relative strengths but also their ability to interfere with another promoter when present in the same transient expression system. This also has implications for their use in multi- gene engineering strategies if there is a need for controlled/tunable expression of multiple genes. The work with ATF6 and ERSE showed how ‘promoter interference’ could be used to our advantage and not necessarily be just a negative occurrence. One hypothesis for why promoter interference occurs is there is competition between promoters for shared transcription factors (TFs). The promoters were analysed for potential transcription factor binding sites (TFBSs) using the programs MatInspector and ModelInspector. The analysis showed that the SV40 promoter had the least number and variety of potential TFBSs. Both the CMV and EF1α had greater numbers and variety of potential TFBSs. All three promoters had common potential TFBSs but the SV40 promoter shared a greater proportion of its sites with the other two promoters. The number of potential TFBSs and the proportion which were shared reflected both the strength and the ability of a promoter to interfere with another. All three contained TFBSs for the SP1 (specificity protein 1) family of TFs and over- expression of SP1 counter-acted the effects of promoter interference showing that it can affect the expression of all three promoters. However, promoter interference will involve more than just a single TF and also more than just competition for transcriptional activators. This is the first time these three promoters have been compared in terms of the potential TFBSs they contain. The TFBS analysis highlighted the complexity in the control of these promoters and with the effects of promoter interference means that they will be ill suited for the controlled expression of multiple genes without modification. The work in this thesis was directed towards the controlled expression of multiple genes in mammalian cells for recombinant protein production. We have presented one novel way of controlling the expression of one/two genes with the rest of the thesis looking at the phenomenon of ‘promoter interference’ between three commonly used promoters. This thesis tries to highlight the importance of multi-gene expression systems as well showing that these three promoters may not be suitable without further modification and also the importance of considering promoter interactions when more than one is present in the same system. The switch to completely synthetic multi-gene control systems is something we envisage happening in the future as the complexities and capabilities of these systems grow. 4 Nathan West The University of Sheffield 5 Nathan West The University of Sheffield 6 Nathan West The University of Sheffield Acknowledgements I would first like to thank my supervisor, Prof. David James for giving me the chance to study for a PhD in his lab. I also want to thank him for all his support and advice throughout the duration of my PhD. I am also grateful to the BBSRC for funding me. A special mention has to go to my lab group including all past and present members who made the experience of a PhD so much greater. I wish them all the best of luck with whatever they decide to do in the future and let them know I really treasured their friendship. I would also like to say thank you to all the other PhD students, post-docs, supervisors, technicians and other members of staff in the department who made it enjoyable place to study. A special thanks goes to my friends who don’t work at the department (Rob & Shaun) or live in Sheffield (Gav & Bown). Sorry for all the special occasions that I may have missed due to being stuck in the lab and thank you for understanding and not being angered by my absence. Another thank you goes to the people I started, firstly my Masters and then my PhD with who have become great friends. Leon, thanks for all your help and advice even if I did ask you some stupid questions. Stephen, the climbing sessions were always fun. Joseph, because you put me in your acknowledgements. Steve, for the sessions in the pub and laughs that really helped me unwind and ease the stress. Lastly, I would like to especially thank my parents, Alan and Denise, my grandparents, Joan and Bill and my girlfriend Danying who without your love and support I would never have made it. It was worth it all in the end. ffffffffffffffffffffffffffffffffffffffffffff 7 Nathan West The University of Sheffield 8 Nathan West The University of Sheffield Contents Thesis Abstract ......................................................................................................... 3 Acknowledgements .................................................................................................. 7 Contents ................................................................................................................... 9 Abbreviation List .................................................................................................... 15 Chapter 1 – Introduction and Research Aims/Objectives ......................................... 19 1.1 – Introduction ........................................................................................................ 19 1.2 – Research Aims and Objectives............................................................................ 23 1.2.1 – Aims.............................................................................................................. 23 1.2.2 – Objectives .................................................................................................... 23 Chapter 2 – Literature Review ................................................................................. 25 2.1 – The Development of Biopharmaceutical Production in Mammalian Cells ........ 25 2.1.1 – Introduction ................................................................................................
Recommended publications
  • Supplemental Materials ZNF281 Enhances Cardiac Reprogramming
    Supplemental Materials ZNF281 enhances cardiac reprogramming by modulating cardiac and inflammatory gene expression Huanyu Zhou, Maria Gabriela Morales, Hisayuki Hashimoto, Matthew E. Dickson, Kunhua Song, Wenduo Ye, Min S. Kim, Hanspeter Niederstrasser, Zhaoning Wang, Beibei Chen, Bruce A. Posner, Rhonda Bassel-Duby and Eric N. Olson Supplemental Table 1; related to Figure 1. Supplemental Table 2; related to Figure 1. Supplemental Table 3; related to the “quantitative mRNA measurement” in Materials and Methods section. Supplemental Table 4; related to the “ChIP-seq, gene ontology and pathway analysis” and “RNA-seq” and gene ontology analysis” in Materials and Methods section. Supplemental Figure S1; related to Figure 1. Supplemental Figure S2; related to Figure 2. Supplemental Figure S3; related to Figure 3. Supplemental Figure S4; related to Figure 4. Supplemental Figure S5; related to Figure 6. Supplemental Table S1. Genes included in human retroviral ORF cDNA library. Gene Gene Gene Gene Gene Gene Gene Gene Symbol Symbol Symbol Symbol Symbol Symbol Symbol Symbol AATF BMP8A CEBPE CTNNB1 ESR2 GDF3 HOXA5 IL17D ADIPOQ BRPF1 CEBPG CUX1 ESRRA GDF6 HOXA6 IL17F ADNP BRPF3 CERS1 CX3CL1 ETS1 GIN1 HOXA7 IL18 AEBP1 BUD31 CERS2 CXCL10 ETS2 GLIS3 HOXB1 IL19 AFF4 C17ORF77 CERS4 CXCL11 ETV3 GMEB1 HOXB13 IL1A AHR C1QTNF4 CFL2 CXCL12 ETV7 GPBP1 HOXB5 IL1B AIMP1 C21ORF66 CHIA CXCL13 FAM3B GPER HOXB6 IL1F3 ALS2CR8 CBFA2T2 CIR1 CXCL14 FAM3D GPI HOXB7 IL1F5 ALX1 CBFA2T3 CITED1 CXCL16 FASLG GREM1 HOXB9 IL1F6 ARGFX CBFB CITED2 CXCL3 FBLN1 GREM2 HOXC4 IL1F7
    [Show full text]
  • Deletion of Vax1 from Gonadotropin-Releasing Hormone (Gnrh) Neurons Abolishes Gnrh Expression and Leads to Hypogonadism and Infertility
    3506 • The Journal of Neuroscience, March 23, 2016 • 36(12):3506–3518 Cellular/Molecular Deletion of Vax1 from Gonadotropin-Releasing Hormone (GnRH) Neurons Abolishes GnRH Expression and Leads to Hypogonadism and Infertility Hanne M. Hoffmann,1 Crystal Trang,1 Ping Gong,1 Ikuo Kimura,2 Erica C. Pandolfi,1 and XPamela L. Mellon1 1Department of Reproductive Medicine and the Center for Reproductive Science and Medicine, University of California, San Diego, La Jolla, California 92093-0674, and 2Department of Applied Biological Science, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu-shi 183-8509, Japan Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are at the apex of the hypothalamic-pituitary-gonadal axis that regu- lates mammalian fertility. Herein we demonstrate a critical role for the homeodomain transcription factor ventral anterior homeobox 1 (VAX1) in GnRH neuron maturation and show that Vax1 deletion from GnRH neurons leads to complete infertility in males and females. Specifically, global Vax1 knock-out embryos had normal numbers of GnRH neurons at 13 d of gestation, but no GnRH staining was detected by embryonic day 17. To identify the role of VAX1 specifically in GnRH neuron development, Vax1flox mice were generated and lineage tracing performed in Vax1flox/flox:GnRHcre:RosaLacZ mice. This identified VAX1 as essential for maintaining expression of Gnrh1. The absence of GnRH staining in adult Vax1flox/flox:GnRHcre mice led to delayed puberty, hypogonadism, and infertility. To address the mechanism by which VAX1 maintains Gnrh1 transcription, the capacity of VAX1 to regulate Gnrh1 transcription was evaluated in the GnRH cell lines GN11 and GT1-7.
    [Show full text]
  • Lncrna Mediated Hijacking of T-Cell Hypoxia Response Pathway by Mycobacterium Tuberculosis Predicts Latent to Active Progression in Humans
    bioRxiv preprint doi: https://doi.org/10.1101/2020.04.11.037176; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. lncRNA Mediated Hijacking of T-cell Hypoxia Response Pathway by Mycobacterium Tuberculosis Predicts Latent to Active Progression in Humans 1# 1# 2# 1* Jyotsana Mehra ,​ Vikram Kumar ,​ Priyansh Srivastava ,​ Tavpritesh Sethi ​ ​ ​ ​ 1 1 2 1* [email protected] ,​ [email protected] ,​ [email protected] ,​ [email protected] ​ ​ ​ ​ ​ ​ ​ ​ 1 2 Indraprastha​ Institute of Information Technology, Delhi, India, Amity​ University, Noida, Uttar Pradesh, India ​ # * Equal​ Contribution, Corresponding​ Author ​ ​ Abstract- Cytosolic functions of Long non-coding RNAs including mRNA translation masking and ​ sponging are major regulators of biological pathways. Formation of T cell- bounded hypoxic granuloma is a host immune defense for containing infected Mtb-macrophages. Our study exploits the mechanistic pathway of Mtb-induced HIF1A silencing by the antisense lncRNA-HIF1A-AS2 in T cells. Computational analysis of in-vitro T-cell stimulation assays in progressors(n=119) versus latent(n=221) tuberculosis patients revealed the role of lncRNA mediated disruption of hypoxia adaptation pathways in progressors. We found 291 upregulated and 227 downregulated lncRNAs that were correlated at mRNA level with HIF1A and HILPDA which are major players in hypoxia response. We also report novel lncRNA-AC010655 (AC010655.4 and AC010655.2) in cis with HILPDA, both of which contain binding sites for the BARX2 transcription factor, thus indicating a mechanistic role.
    [Show full text]
  • Original Article the Association Between the EPHA3 Gene Polymorphism and Non-Syndromic Cleft Lip with Or Without Palate in the Western Han Chinese Population
    Int J Clin Exp Med 2020;13(9):7060-7070 www.ijcem.com /ISSN:1940-5901/IJCEM0112790 Original Article The association between the EPHA3 gene polymorphism and non-syndromic cleft lip with or without palate in the western Han Chinese population Yulan Zhou1*, Jiayu Shi2*, Bing Shi1, Zhonglin Jia1 1State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Department of Cleft Lip and Palate, West China Hospital of Stomatology, Sichuan University, Chengdu, China; 2Division of Growth and Development and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, USA. *Equal contributors. Received April 29, 2020; Accepted July 17, 2020; Epub September 15, 2020; Published September 30, 2020 Abstract: Non-syndromic cleft lip with or without palate (NSCL/P) is a common craniofacial congenital disease which results from multiple susceptibility genes and adverse environmental factors. A recent study reported that the T al- lele of rs7650466 of the EPHA3 gene is a genetic risk factor in the etiology of NSCL/P among the northern Chinese Han population. This study aimed to evaluate the association between the EPHA3 gene variations and NSCL/P in the western Han Chinese population. Here, we conducted targeted region sequencing around rs7632427 (EPHA3) among 159 unrelated NSCL/P cases from the western Han Chinese population and performed a gene-based burden analysis on the rare variations and a single variation association analysis on the common SNVs (single nucleotide variants). Then we found 438 SNVs and 120 indels in all. A burden analysis showed no statistical signifi- cance. The association analysis results suggested that the common SNV rs13094064 was associated with NSCL/P (P=1.86E-07 and OR=0.17), and rs7632427 was most closely related to NSCLO (P=7.49E-05 and OR=0.315).
    [Show full text]
  • University of Dundee Molecular Screening of VAX1 Gene
    University of Dundee Molecular Screening of VAX1 Gene Polymorphisms Uncovered the Genetic Heterogeneity of Non-Syndromic Orofacial Cleft in Saudi Arabian Patients Jafar Sabbagh, Heba ; Innes, Nicola; Edris Ahmed, Sherif ; Butali, Azeez; Abdulbaset Alnamnakani, Eman ; Rabah, Sari Published in: Genetic Testing and Molecular Biomarkers DOI: 10.1089/gtmb.2018.0207 Publication date: 2019 Document Version Peer reviewed version Link to publication in Discovery Research Portal Citation for published version (APA): Jafar Sabbagh, H., Innes, N., Edris Ahmed, S., Butali, A., Abdulbaset Alnamnakani, E., Rabah, S., Hamdan, M. A., Alhamlan, N., Abdulhameed, F. D., Hassan, M. H. A., Bassam Al Mahdi, H., Alamoudi, N. M., Alaki, S. M., & Mossey, P. (2019). Molecular Screening of VAX1 Gene Polymorphisms Uncovered the Genetic Heterogeneity of Non-Syndromic Orofacial Cleft in Saudi Arabian Patients. Genetic Testing and Molecular Biomarkers, 23(1), 45- 50. https://doi.org/10.1089/gtmb.2018.0207 General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from Discovery Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
    [Show full text]
  • Clinical and Molecular Investigation of Rare
    CLINICAL AND MOLECULAR INVESTIGATION OF RARE CONGENITAL DEFECTS OF THE PALATE RIMANTE SESELGYTE A thesis submitted for the degree of Doctor of Philosophy to University College London August 2019 page Title 1 DECLARATION I, Rimante Seselgyte, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed……………………….. Declaration 3 ABSTRACT Cleft palate (CP) affects around 1/1500 live births and, along with cleft lip, is one of the most common forms of birth defect. The studies presented here focus on unusual defects of the palate, especially to understand better the rarely reported but surprisingly common condition called submucous cleft palate (SMCP). The frequency and consequences of SMCP from a surgical perspective were first investigated based on the caseload of the North Thames Cleft Service at Great Ormond Street Hospital and St Andrew's Centre, Broomfield Hospital, Mid Essex Hospitals Trust. It was previously reported that up to 80% of individuals with unrepaired SMCP experience speech difficulties as a consequence of velopharyngeal insufficiency (VPI). Attempted repair of the palatal defect can sometimes give poor results, so controversies still exist about the correct choice of surgical technique to use. Over 23 years, 222 patients at The North Thames Cleft Service underwent operations to manage SMCP. Nearly half of them (42.8%) were diagnosed with 22q11.2 deletion syndrome (22q11.2 DS). The first operation was palate repair, with an exception of one case, followed by a second surgical intervention required in approximately half of the patients.
    [Show full text]
  • Regulatory Interactions Between Vax1, Pax6, and Mir-7 Regionalize the Lateral Ventricular-Subventricular Zone During Post-Natal Olfactory Bulb Neurogenesis in Mice
    bioRxiv preprint doi: https://doi.org/10.1101/2020.04.24.059428; this version posted April 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Regulatory interactions between Vax1, Pax6, and miR-7 regionalize the lateral Ventricular-Subventricular Zone during post-natal Olfactory Bulb neurogenesis in mice Nathalie Coré 1, Andrea Erni 1, Pamela L. Mellon 2, Hanne M. Hoffmann 2, Christophe Béclin 1 and Harold Cremer 1 1. Aix Marseille University, CNRS, IBDM, Campus de Luminy, Marseille, France 2. Department of Obstetrics, Gynecology, and Reproductive Sciences and the Center for Reproductive Science and Medicine, University of California, San Diego, La Jolla, California 92093-0674, USA For correspondence: [email protected] and [email protected] Abstract Several subtypes of interneurons destined for the olfactory bulb are continuously generated after birth by neural stem cells located in the ventricular-subventricular zones of the lateral ventricles. Future neuronal identity depends on the positioning of pre-determined neural stem cells along the ventricle walls, which, in turn, depends on delimited expression domains of transcription factors and their cross regulatory interactions. However, mechanisms underlying positional identity of neural stem cells are still poorly understood. Here we show that the transcription factor Vax1 controls the production of two specific neuronal sub-types. First, it is directly necessary to generate Calbindin expressing interneurons from ventro-lateral progenitors. Second, it represses the generation of dopaminergic neurons by dorso-lateral progenitors through inhibiting Pax6 expression in the dorso-lateral wall.
    [Show full text]
  • The LIM Protein Complex Establishes a Retinal Circuitry of Visual Adaptation
    RESEARCH ARTICLE The LIM protein complex establishes a retinal circuitry of visual adaptation by regulating Pax6 a-enhancer activity Yeha Kim1, Soyeon Lim1†, Taejeong Ha1†, You-Hyang Song1, Young-In Sohn1, Dae-Jin Park2, Sun-Sook Paik3, Joo-ri Kim-Kaneyama4, Mi-Ryoung Song5, Amanda Leung6, Edward M Levine6, In-Beom Kim3, Yong Sook Goo2, Seung-Hee Lee1, Kyung Hwa Kang7, Jin Woo Kim1* 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, South Korea; 2Department of Physiology, Chungbuk National University School of Medicine, Cheongju, South Korea; 3Department of Anatomy, College of Medicine, The Catholic University of Korea, Seoul, South Korea; 4Department of Biochemistry, Showa University School of Medicine, Tokyo, Japan; 5Department of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, South Korea; 6Department of Ophthalmology and Visual Sciences, Vanderbilt University, Nashville, United States; 7KAIST Institute of BioCentury, Daejeon, South Korea Abstract The visual responses of vertebrates are sensitive to the overall composition of retinal interneurons including amacrine cells, which tune the activity of the retinal circuitry. The expression of Paired-homeobox 6 (PAX6) is regulated by multiple cis-DNA elements including the intronic a- *For correspondence: enhancer, which is active in GABAergic amacrine cell subsets. Here, we report that the [email protected] transforming growth factor ß1-induced transcript 1 protein (Tgfb1i1) interacts with the LIM domain transcription factors Lhx3 and Isl1 to inhibit the a-enhancer in the post-natal mouse retina. †These authors contributed Tgfb1i1-/- mice show elevated a-enhancer activity leading to overproduction of Pax6DPD isoform equally to this work that supports the GABAergic amacrine cell fate maintenance.
    [Show full text]
  • Identification of Shared and Unique Gene Families Associated with Oral
    International Journal of Oral Science (2017) 9, 104–109 OPEN www.nature.com/ijos ORIGINAL ARTICLE Identification of shared and unique gene families associated with oral clefts Noriko Funato and Masataka Nakamura Oral clefts, the most frequent congenital birth defects in humans, are multifactorial disorders caused by genetic and environmental factors. Epidemiological studies point to different etiologies underlying the oral cleft phenotypes, cleft lip (CL), CL and/or palate (CL/P) and cleft palate (CP). More than 350 genes have syndromic and/or nonsyndromic oral cleft associations in humans. Although genes related to genetic disorders associated with oral cleft phenotypes are known, a gap between detecting these associations and interpretation of their biological importance has remained. Here, using a gene ontology analysis approach, we grouped these candidate genes on the basis of different functional categories to gain insight into the genetic etiology of oral clefts. We identified different genetic profiles and found correlations between the functions of gene products and oral cleft phenotypes. Our results indicate inherent differences in the genetic etiologies that underlie oral cleft phenotypes and support epidemiological evidence that genes associated with CL/P are both developmentally and genetically different from CP only, incomplete CP, and submucous CP. The epidemiological differences among cleft phenotypes may reflect differences in the underlying genetic causes. Understanding the different causative etiologies of oral clefts is
    [Show full text]
  • Review Article Association Between IRF6 Rs642961 Polymorphism and Non-Syndromic Cleft Lip with Or Without a Cleft Palate: a Systematic Review and Meta-Analysis
    Int J Clin Exp Med 2018;11(4):3033-3042 www.ijcem.com /ISSN:1940-5901/IJCEM0066579 Review Article Association between IRF6 rs642961 polymorphism and non-syndromic cleft lip with or without a cleft palate: a systematic review and meta-analysis Tzu-Hui Lee1*, Tsung-Ta Liu2*, Chih-Wei Sung3,4*, Ju-Chi Ou5, Jui-Ju Yeh6, Yung-Ting Kuo1,7, Yuan-Hung Wang8,9 Departments of 1Pediatrics, 5Emergency Medicine, 6Family Medicine, 8Medical Research, Shuang Ho Hospital, Taipei Medical University, New Taipei City 23561, Taiwan; 2Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan; 3Department of Emergency Medicine, National Taiwan Univer- sity Hospital Hsin-Chu Branch, Hsinchu, Taiwan; 4Department of Emergency Medicine, National Taiwan University Hospital, Taipei, Taiwan; 7Department of Pediatrics, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan; 9Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan. *Equal contributors. Received October 2, 2017; Accepted January 29, 2018; Epub April 15, 2018; Published April 30, 2018 Abstract: Non-syndromic cleft lip with or without a cleft palate (NSCL/P) is one of the most common newborn malformations. sPrevious studies have reported that genetic variations of interferon regulatory factor 6 (IRF6) poly- morphisms are associated with NSCL/P. However, the effect sizes of individual studies still vary. Our present study is a meta-analysis to investigate the association of IRF6 rs642961 polymorphism with NSCL/P. A literature search in PubMed was performed to select eligible literatures including observational studies which evaluated association between IRF6 polymorphisms and NSCL/P.
    [Show full text]
  • Using the Mouse Retina to Model the Role of Sox2 in Neural Induction
    USING THE MOUSE RETINA TO MODEL THE ROLE OF SOX2 IN NEURAL INDUCTION Whitney E. Heavner A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for a degree of Doctor of Philosophy in the Program in Genetics and Molecular Biology Chapel Hill 2013 Approved by Robert Duronio, Ph.D. Stephen Crews, Ph.D. Norman Sharpless, M.D. Ellen Weiss, Ph.D. Mark Zylka, Ph.D. © 2013 Whitney E. Heavner ALL RIGHTS RESERVED ii ABSTRACT WHITNEY E. HEAVNER: Using the Mouse Retina to Model the Role of SOX2 in Neural Induction (Under the direction of Dr. Larysa Pevny) Neural competence is the ability of a progenitor cell to generate a neuron. The eye is one of the few tissues derived from the neural ectoderm that contains both neurogenic and non-neurogenic cells, all of which arise from a common progenitor pool. Therefore, the eye is a particularly useful model to study the molecular mechanisms that confer neural competence. Moreover, this cell fate dichotomy is highly reminiscent of the earlier process of neural induction, or the decision of an ectoderm precursor cell to become neural plate or epidermis. The HMG-box transcription factor SOX2 is crucial for both of these processes. Little is known about the role of SOX2 in neural induction, and what is known has been worked out primarily in lower vertebrates. Humans and mice with mutations in SOX2 exhibit a range of neural defects; therefore, from the perspective of human health, it is important to understand SOX2’s function in mammalian neuroepithelium.
    [Show full text]
  • TFAP2 Transcription Factors Are Regulators of Lipid Droplet Biogenesis
    bioRxiv preprint doi: https://doi.org/10.1101/282376; this version posted March 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 TFAP2 transcription factors are 2 regulators of lipid droplet biogenesis 3 4 5 Cameron C Scott1, Stefania Vossio1, Jacques Rougemont2, Jean Gruenberg1,3 6 7 8 9 10 11 12 1. Department of Biochemistry, University of Geneva, 30 quai Ernest Ansermet 13 1211 Geneva 4, Switzerland 14 2. Department of Theoretical Physics, University of Geneva, 24 quai Ernest Ansermet 15 1211 Geneva 4, Switzerland 16 17 3. To whom correspondence should be addressed: [email protected] 18 19 20 21 22 1 bioRxiv preprint doi: https://doi.org/10.1101/282376; this version posted March 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Abstract 2 How trafficking pathways and organelle abundance adapt in response to metabolic 3 and physiological changes is still mysterious, although a few transcriptional regulators of 4 organellar biogenesis have been identified in recent years. We previously found that the Wnt 5 signaling directly controls lipid droplet formation, linking the cell storage capacity to the 6 established functions of Wnt in development and differentiation. In the present paper, we 7 report that Wnt-induced lipid droplet biogenesis does not depend on the canonical TCF/LEF 8 transcription factors.
    [Show full text]