JoVE Journal > Genetics
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
Referências
Tadução automática
Please note that all translations are automatically generated. Click here for the English version.
Genética

血管动力尾静脉注射对小鼠肝细胞基因的本构和诱导系统的修饰

Published: February 02, 2018
doi:
10.3791/56613
, , , , ,
1Department of Medicine II, Klinikum rechts der Isar,Technische Universität München, 2Department of Pneumology, Center for Medicine,Medical Center University of Freiburg

Summary

水动力尾静脉注射转的融合载体, 可以稳定转染小鼠肝细胞在体内.在这里, 我们提出了一个实用的转染系统的协议, 使一个单一的转基因或联合本构和强力霉素诱导表达的转基因或和平 shRNA 在肝脏的长期本构表达。

Abstract

在肝癌的研究模式, 再生, 炎症和纤维化, 灵活的系统为体内基因表达和沉默是非常有用的。以转为基础的水动力尾静脉注射是一种有效的成年小鼠肝细胞遗传操作方法。除了本构式转基因表达外, 该系统还可用于更先进的应用, 如 shRNA 介导的基因敲除, CRISPR/Cas9 系统诱导基因突变或诱导系统的暗示。将本构 CreER 表达与转基因或 shRNA 选择的诱导表达结合起来, 作为这一技术的一个例子。我们涵盖了多步骤的过程, 从准备的睡美人-转结构, 注射和治疗小鼠, 并准备肝组织进行分析的免疫。该系统是实现肝细胞复杂遗传操作的可靠而有效的方法。它是特别有用的结合与 loxP 的小鼠品系, 可用于各种模型的研究肝病。

Introduction

慢性肝病是一个主要的健康负担全球1。动物研究模型是肝脏疾病研究的重要工具, 有助于解答肝脏再生、肝脏炎症、脂肪以及肝癌2等复杂问题。大量的这些动物模型依赖于肝细胞的基因修饰。因此, 有效的工具, 操纵基因表达在肝细胞是有益的3。建立的方法, 如育种的基因工程小鼠菌株或病毒载体的生成肝细胞感染要么费时, 港口安全关注, 或屈服于肝细胞的不良转基因表达在体内4,5. 水动力尾静脉注射 (HTVI) 是一种替代方法的体内转染肝细胞允许方便, 快速, 和经济高效的基因功能的审讯。对于 HTVI, 携带所需 DNA 序列的载体被溶解在相当于注射动物体重10% 的盐水量中。然后在5-10 秒6内将该解决方案注入尾部静脉。超过心脏输出, 盐水从下腔静脉流入肝静脉, 导致肝脏扩张和肝细胞的水动力转染7。为了实现稳定的基因组集成, 该方法已与基于转的矢量 (如休眠美人-转系统) 相结合。该系统介导的重组的目标载体与基因组重组网站的催化睡美人-转8,9。对于肝纤维化或癌变的模型, 在疾病模型的某些时间点过度或沉默基因往往是可取的。为此, 可诱导基因表达的工具, 如 LoxP 系统或四环素诱导基因表达系统 (春节) 可以使用10。

在这里, 我们描述了一个协议的在体内转染小鼠肝细胞使用 HTVI 的睡美人转的系统。除了一个在肝脏特异启动子控制下的转基因的稳定、本构表达的协议外, 我们还描述了一种更先进的向量系统, 它将本构型他莫昔芬重组 (CreER) 表达与可诱导表达的转基因或 rna 适应的 shRNA (和平 shRNA), 称为 pTC 的春节系统11。在这个向量系统中, 可诱导的转基因或和平 shRNAs 为四环素相关的表达被克隆到骨干载体与重组克隆系统, 允许快速和容易生成新的载体12。本视频指南包括制备合适的载体, 注射和治疗小鼠, 以实现诱导转基因/和平 shRNA 表达, 最后准备肝脏组织进行分析。本协议中描述的方法旨在使任何选择的基因 loxP 介导的小鼠系统的表达或击倒, 使得它成为一种广泛适用的肝病研究系统。

Protocol

所有动物实验都是根据实验室动物的护理和使用指南进行的, 并得到主管当局的批准 (Regierung Oberbayern、慕尼黑、德国和斯坦福机构动物保育和使用委员会,斯坦福, 加利福尼亚, 美国)。在补充表 S1 中提供了所有用于克隆的质粒的清单 (步骤1至 4)。 1. 本构基因表达的转基因克隆 设计引物为转基因放大13,14。 将总署 (TTAATT…

Representative Results

水动力尾静脉注射转染效果:单次注射转染水力的小鼠肝细胞百分比是可变的, 取决于注射量、注射时间、注入 DNA 量和注入结构的大小等多个参数6, 22,23。此外, 转染效率一般较低, 在较大的动物, 较大的血管直径, 以及较大的正弦面积导致整体压力下降。为了直观地显示转染效率, CreER 转结?…

Discussion

水动力尾静脉注入肝细胞的转染已成为15年前6以来建立的一种方法。注入的体积超过心脏输出, 从下腔静脉流向肝脏的窦7, 导致转染约 10-20%, 在某些情况下多达40% 的肝细胞25,26。成功转染的预测因子是每次注入时间的注入量22,23。因此, 低转染效率 (图 1A, 左面板…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到德国德意志 Krebshilfe (111289 号至 ue)、露西尔的儿童健康基金会的支持 (欧内斯特和阿米莉亚-奖授予博士后奖学金-UL1 RR025744 至 ue)。我们感谢马克. 凯博士的向量结构和实验建议和朱利安博士鼠和实验支持。

Materials

General Material
GeneRuler 1 kb Plus DNA Ladder Thermo Fisher #SM1331 DNA ladder for electrophoresis
Tissue-Tek O.C.T. Sakura 4583 embedding of cryo-sections
Biozym LE Agarose Biozym 840004
Ethidium bromide Sigma-Aldrich E7637-1G
D(+)-Saccharose Carl Roth 4621.1 For sweetening of the doxycyline solution
Ampicillin Sodium Salt AppliChem A0839,0010 For selection of Amp-resistant clones
LB Agar (Luria/Miller) Carl Roth X969.1
LB Broth (Luria/Miller) Carl Roth X968.1
S.O.C. Medium Thermo Fischer 15544034
Gentamicin sulfate AppliChem A1492,0001 For selection of Gentamicin-resistant clones
Roti-Histofix 4 % Fa. Roth P087.6 para-formaldehyde solution
T4 DNA Ligase New England BioLabs M0202S
GatewayTM LR ClonaseTM II Enzyme Mix invitrogen/ThermoFisher 11791-020 contains LR-clonase enzyme mix II and proteinase K
DB3.1 Competent Cells Thermo Fisher 11782-018
Stbl3 Chemically Competent E. coli Thermo Fisher C737303
Name Company Catalog Number Comments
Restriction Enzymes
PacI New England BioLabs R0547S
AscI New England BioLabs R0558S
FseI New England BioLabs R0588S
SacI New England BioLabs R0156S
SpeI New England BioLabs R0133S
KpnI New England BioLabs R0142S
NotI New England BioLabs R0189S
XhoI New England BioLabs R0146S
BfuAI New England BioLabs R0701S
Name Company Catalog Number Comments
Kits
QIAquick Gel Extraction Kit Qiagen 28704 For DNA Extraction from gel
NucleoSpin Gel and PCR Clean Up Macherey & Nagel 740609.10
NucleoBond PC20 Macherey & Nagel 740571 Plasmid extraction (Mini prep)
NucleoBond PC500 Macherey & Nagel 740574 Plasmid extraction (Maxi prep)
Phusion High-Fidelity DNA Polymerase Thermo Fisher F530S
Name Company Catalog Number Comments
Materials for Mouse Experiments
Injekt Syringe F 1 ml Braun 9166017V For intraperitoneal injection
Omnifix Luer 3 ml Braun 4616025V For intravenous injection
Sterican Cannula 24G Braun 4657675
Sterican Cannula 27G Braun 4657705
Tamoxifen Sigma-Aldrich T5648-1G For CreER activation
Corn oil Sigma-Aldrich C8267-500ML Carrier for tamoxifen injections
Doxycycline hyclate AppliChem A2951,0025 Activation of tetracycline-dependent expression
Injekt 10 ml Syringe Braun 4606108V
Filtropur S 0.2 Sarstedt 831,826,001 For filtration of doxycycline
NaCl 0,9% Braun 3200905 Carrier for intravenous injections
Falcon Conical Tube 50ml Corning Life Science 352095
Infrared Lamp N/A N/A For warming of mouse tail
IVIS Perkin Elmer 124262 In vivo imaging system
Name Company Catalog Number Comments
Plasmids for cloning of sleeping beauty-transposon vectors for HTVI.
pTC n/a Vector for constitutive gene expression, ref. 15
pEN_TTmcs Addgene #25755 Entry vector for inducible gene expression, ref. 19
pEN_TTGmiRc2 Addgene #25753 Entry vector for inducible miR-shRNA expression with co-expression of GFP, ref. 19
pEN_TTmiRc2 Addgene #25752 Entry vector for inducible miR-shRNA expression without co-expression of GFP, ref. 19
pTC ApoE-Tet Addgene #85578 Expression vector for inducible gene or miR-shRNA expression with ApoE.HCR.hAAT promotor, ref. 11
pTC-CMV-Tet Addgene #85577 Expression vector for inducible gene or miR-shRNA expression with CMV promotor, ref. 11
DOWNLOAD MATERIALS LIST

Referências

  1. Byass, P. The global burden of liver disease: a challenge for methods and for public health. BMC Med. 12, 159 (2014).
  2. Liu, Y., et al. Animal models of chronic liver diseases. Am J Physiol Gastrointest Liver Physiol. 304 (5), G449-G468 (2013).
  3. Frese, K. K., Tuveson, D. A. Maximizing mouse cancer models. Nat Rev Cancer. 7 (9), 645-658 (2007).
  4. Dow, L. E., et al. Conditional reverse tet-transactivator mouse strains for the efficient induction of TRE-regulated transgenes in mice. PLoS One. 9 (4), e95236 (2014).
  5. Lundstrom, K. Latest development in viral vectors for gene therapy. Trends Biotechnol. 21 (3), 117-122 (2003).
  6. Liu, F., Song, Y., Liu, D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 6 (7), 1258-1266 (1999).
  7. Kanefuji, T., et al. Hemodynamics of a hydrodynamic injection. Mol Ther Methods Clin Dev. 1, 14029 (2014).
  8. Ivics, Z., Izsvak, Z. Sleeping Beauty Transposition. Microbiol Spectr. 3 (2), (2015).
  9. Hausl, M. A., et al. Hyperactive sleeping beauty transposase enables persistent phenotypic correction in mice and a canine model for hemophilia B. Mol Ther. 18 (11), 1896-1906 (2010).
  10. Doyle, A., McGarry, M. P., Lee, N. A., Lee, J. J. The construction of transgenic and gene knockout/knockin mouse models of human disease. Transgenic Res. 21 (2), 327-349 (2012).
  11. Hubner, E. K., et al. An in vivo transfection system for inducible gene expression and gene silencing in murine hepatocytes. J Gene Med. 19 (1-2), (2017).
  12. Katzen, F. Gateway((R)) recombinational cloning: a biological operating system. Expert Opin Drug Discov. 2 (4), 571-589 (2007).
  13. Chuang, L. Y., Cheng, Y. H., Yang, C. H. Specific primer design for the polymerase chain reaction. Biotechnol Lett. 35 (10), 1541-1549 (2013).
  14. Lorenz, T. C. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp. (63), e3998 (2012).
  15. Ehmer, U., et al. Organ size control is dominant over Rb family inactivation to restrict proliferation in vivo. Cell Rep. 8 (2), 371-381 (2014).
  16. Froger, A., Hall, J. E. Transformation of plasmid DNA into E. coli using the heat shock method. J Vis Exp. (6), e253 (2007).
  17. Sanger, F., Coulson, A. R. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol. 94 (3), 441-448 (1975).
  18. Koontz, L. Explanatory chapter: how plasmid preparation kits work. Methods Enzymol. 529, 23-28 (2013).
  19. Shin, K. J., et al. A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. Proc Natl Acad Sci U S A. 103 (37), 13759-13764 (2006).
  20. Hubner, E. K., et al. An in vivo transfection system for inducible gene expression and gene silencing in murine hepatocytes. J Gene Med. , (2016).
  21. Yant, S. R., Huang, Y., Akache, B., Kay, M. A. Site-directed transposon integration in human cells. Nucleic Acids Res. 35 (7), e50 (2007).
  22. Zhang, G., Budker, V., Wolff, J. A. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther. 10 (10), 1735-1737 (1999).
  23. Maruyama, H., et al. High-level expression of naked DNA delivered to rat liver via tail vein injection. J Gene Med. 4 (3), 333-341 (2002).
  24. Bell, J. B., Aronovich, E. L., Schreifels, J. M., Beadnell, T. C., Hackett, P. B. Duration of expression and activity of Sleeping Beauty transposase in mouse liver following hydrodynamic DNA delivery. Mol Ther. 18 (10), 1796-1802 (2010).
  25. Brunetti-Pierri, N., Lee, B. Gene therapy for inborn errors of liver metabolism. Mol Genet Metab. 86 (1-2), 13-24 (2005).
  26. Atta, H. M. Gene therapy for liver regeneration: experimental studies and prospects for clinical trials. World J Gastroenterol. 16 (32), 4019-4030 (2010).
  27. Malato, Y., et al. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration. J Clin Invest. 121 (12), 4850-4860 (2011).
  28. Weber, J., et al. CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice. Proc Natl Acad Sci U S A. 112 (45), 13982-13987 (2015).
  29. Viecelli, H. M., et al. Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver. Hepatology. 60 (3), 1035-1043 (2014).
  30. Delerue, F., White, M., Ittner, L. M. Inducible, tightly regulated and non-leaky neuronal gene expression in mice. Transgenic Res. 23 (2), 225-233 (2014).
  31. Izsvak, Z., Ivics, Z., Plasterk, R. H. Sleeping Beauty, a wide host-range transposon vector for genetic transformation in vertebrates. J Mol Biol. 302 (1), 93-102 (2000).
  32. Koponen, J. K., et al. Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo. Gene Ther. 10 (6), 459-466 (2003).
  33. Saitoh, Y., et al. Dose-dependent doxycycline-mediated adrenocorticotropic hormone secretion from encapsulated Tet-on proopiomelanocortin Neuro2A cells in the subarachnoid space. Hum Gene Ther. 9 (7), 997-1002 (1998).
  34. Yao, M., et al. Conditional Inducible Triple-Transgenic Mouse Model for Rapid Real-Time Detection of HCV NS3/4A Protease Activity. PLoS One. 11 (3), e0150894 (2016).
  35. Santacatterina, F., et al. Down-regulation of oxidative phosphorylation in the liver by expression of the ATPase inhibitory factor 1 induces a tumor-promoter metabolic state. Oncotarget. 7 (1), 490-508 (2016).
  36. Hojman, P., Eriksen, J., Gehl, J. Tet-On induction with doxycycline after gene transfer in mice: sweetening of drinking water is not a good idea. Anim Biotechnol. 18 (3), 183-188 (2007).
  37. Cawthorne, C., Swindell, R., Stratford, I. J., Dive, C., Welman, A. Comparison of doxycycline delivery methods for Tet-inducible gene expression in a subcutaneous xenograft model. J Biomol Tech. 18 (2), 120-123 (2007).
  38. Yuan, L., et al. An HBV-tolerant immunocompetent model that effectively simulates chronic hepatitis B virus infection in mice. Exp Anim. , (2016).
  39. Zhou, Q., et al. RPB5-Mediating Protein Suppresses Hepatitis B Virus (HBV) Transcription and Replication by Counteracting the Transcriptional Activation of Hepatitis B virus X Protein in HBV Replication Mouse Model. Jundishapur J Microbiol. 8 (9), e21936 (2015).
  40. Yagai, T., Miyajima, A., Tanaka, M. Semaphorin 3E secreted by damaged hepatocytes regulates the sinusoidal regeneration and liver fibrosis during liver regeneration. Am J Pathol. 184 (8), 2250-2259 (2014).
  41. Tordella, L., et al. SWI/SNF regulates a transcriptional program that induces senescence to prevent liver cancer. Genes Dev. 30 (19), 2187-2198 (2016).
  42. Ogata, K., Selvaraj, S. R., Miao, H. Z., Pipe, S. W. Most factor VIII B domain missense mutations are unlikely to be causative mutations for severe hemophilia A: implications for genotyping. J Thromb Haemost. 9 (6), 1183-1190 (2011).
  43. Vercellotti, G. M., et al. H-ferritin ferroxidase induces cytoprotective pathways and inhibits microvascular stasis in transgenic sickle mice. Front Pharmacol. 5, 79 (2014).
  44. Ando, M., et al. Prevention of adverse events of interferon gamma gene therapy by gene delivery of interferon gamma-heparin-binding domain fusion protein in mice. Mol Ther Methods Clin Dev. 1, 14023 (2014).
  45. Watcharanurak, K., et al. Regulation of immunological balance by sustained interferon-gamma gene transfer for acute phase of atopic dermatitis in mice. Gene Ther. 20 (5), 538-544 (2013).

Tags

In VivoGenetic ModificationMouse HepatocytesHydrodynamic Tail Vein InjectionConstitutive SystemInducible SystemCreERShRNAVector ConstructLiver ResearchLiver CancerLiver Regeneration
check_url/pt/56613?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Hubner, E. K., Lechler, C., Rösner, T. N., Kohnke-Ertel, B., Schmid, R. M., Ehmer, U. Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection. J. Vis. Exp. (132), e56613, doi:10.3791/56613 (2018).
Baixar citação
Reimpressões e Permissões

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image