Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Summary

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

Abstract

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Introduction

Chronic liver disease presents a major health burden worldwide¹. Animal research models are essential tools in the study of liver disease and have helped to answer complex questions in liver regeneration, hepatic inflammation, and steatosis as well as liver cancer². A substantial number of these animal models rely on the genetic modification of liver cells. Therefore, efficient tools to manipulate gene expression in hepatocytes are helpful³. Established methods such as the breeding of genetically engineered mouse strains or the generation of viral vectors for hepatocyte infection are either time c....

Protocol

All animal experiments were performed according to the guidelines for the care and use of laboratory animals and were approved by responsible authorities (Regierung von Oberbayern, Munich, Germany and Stanford Institutional Animal Care and Use Committee, Stanford, CA, USA). A list of all plasmids for cloning (step 1 through 4) is provided in supplementary table S1.

1. Cloning of a Transgene for Constitutive Gene Expression

1. Design primers for transgene amplification^13,14.
2. Add restriction sites for PacI (TTAATTAA) to the 5' end of the forward primer. Add rest....

Results

Transfection efficacy by hydrodynamic tail vein injection: The percentage of murine hepatocytes that are transfected hydrodynamically by a single injection is variable and depends on multiple parameters such as injection volume, injection time, amount of injected DNA, and size of the injected construct^6,22,23. Additionally, the transfection efficiency is generally lower in larger.......

Discussion

Transfection of hepatocytes with hydrodynamic tail vein injection has become an established method since its introduction more than 15 years ago⁶. The injected volume exceeds cardiac output and flows from the inferior vena cava into the sinusoids of the liver⁷, leading to transfection of about 10-20%, in some cases up to 40% of hepatocytes^25,26. Predictors of a successful transfection are the injected volume per inj.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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