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Method Article

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

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DOI:

10.3791/56613

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February 2nd, 2018

 ,  ,  ,  ,  , 
1Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 2Department of Pneumology, Center for Medicine, Medical Center University of Freiburg

In This Article

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 consuming, harbor safety concerns, or yield poor transgene expression in hepatocytes in vivo4,5. Hydrodynamic tail vein injection (HTVI) is an alternative method for in vivo transfection of hepatocytes allowing for easy, fast, and cost-efficient interrogation of gene function in the liver. For HTVI, a vector carrying the desired DNA sequence is dissolved in a volume of saline corresponding to 10% of the body weight of the injected animal. The solution is then injected into the tail vein within 5-10 s6. Exceeding cardiac output, the saline flows from the inferior vena cava into the liver veins, leading to expansion of the liver and hydrodynamic transfection of hepatocytes7. To achieve stable genomic integration, the method has been combined with transposon-based vectors, such as the sleeping beauty-transposon system. This systems mediates the recombination of target vectors with genomic recombination sites catalyzed by a sleeping beauty-transposase8,9. For models of liver fibrosis or carcinogenesis, it is often desirable to overexpress or silence genes at certain time points of the disease model. For this purpose, tools for inducible gene expression such as the Cre/LoxP-system or the tetracycline-inducible gene expression system (Tet-On) may be used10.

Here, we describe a protocol for in vivo transfection of murine hepatocytes using HTVI of a sleeping beauty transposon-based system. In addition to a protocol for stable, constitutive expression of a transgene under the control of a liver-specific promoter, we describe a more advanced vector system that combines constitutive tamoxifen-dependent Cre recombinase (CreER) expression with the inducible expression of a transgene or microRNA-adapted shRNA (miR-shRNA), called the pTC TET-system11. In this vector system, inducible transgenes or miR-shRNAs for tetracycline-dependent expression are cloned into the backbone vector with a recombinational cloning system, allowing the fast and easy generation of new vectors12. This video-based guide covers the preparation of suitable vectors, injection and treatment of mice to achieve inducible transgene/miR-shRNA expression, and finally preparation of liver tissue for analysis. The method described in this protocol was designed to enable the combination of any Cre/loxP mediated mouse system with the expression or knock-down of any gene of choice, making it a widely applicable system in research of liver disease.

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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 amplification13,14.
  2. Add restriction sites for PacI (TTAATTAA) to the 5' end of the forward primer. Add restriction sites for AscI (GGCGCGCC) or FseI (GGCCGGCC) to the 5' end of the reverse primer.
  3. Amplify transgene by PCR using conditions optimized for the desired transgene14. Purify using a commercially available DNA purification kit.
    NOTE: Annealing time depends on the primers designed in step 1.1., elongation time depends on the length of the construct. For example, for a construct of 1,000 bp length use 60 s of elongation time.
  4. Digest transgene and vector for constitutive gene expression15 with respective restriction nucleases (see step 1.2) and buffer in a total volume of 50 µL at 37 °C overnight. For example, digest construct with 5 units PacI and 5 units FseI if appropriate (see step 1.2).
  5. Gel purify digested vector and insert using a commercial gel extraction kit according to the manufacturer's instructions. Perform a standard ligation of 100 ng of vector and 100–1,000 ng of insert using 400 U T4 ligase at 14 °C for 16 h. Heat inactivate at 65 °C for 10 min.
  6. Transform competent bacteria using a standard heat-shock-protocol16.
  7. Plate on agar plates containing 100 µg/mL ampicillin. Incubate at 30 °C for 24 h.
  8. Pick single colonies, purify plasmid DNA using a commercial miniprep kit according to the manufacturer's instructions and verify the sequence by Sanger sequencing17 (sequencing primer: 5' TGCTGGAGTTCTTCGCC 3').
  9. Use positive colonies for maxi scale amplification of plasmid DNA and purify using an endotoxin-free plasmid preparation kit18 according to the manufacturer's instructions.
  10. Construct is ready for injection, thus continue with step 5.

2. Cloning of a Transgene for Inducible Gene Expression

  1. Design primers for transgene amplification13,14.
  2. Add restriction sites for SacI (GAGCTC), SpeI (ACTAGT), or KpnI (GGTACC) to the 5' end of the forward primer. Add restriction sites for NotI (GCGGCCGC) or XhoI (CTCGAG) to the 5' end of the reverse primer.
  3. Amplify transgene by PCR using conditions optimized for the desired transgene14. Purify using a commercial DNA purification kit.
  4. Digest the purified transgene and 4 µg of Entry vector (pEN_TTmcs-Vector, see Table of Materials)19 separately with appropriate restriction nucleases (see step 2.2) and buffer in a total volume of 50 µL at 37 °C overnight.
  5. Gel purify digested vector and insert using a commercial gel extraction kit according to the manufacturer's instructions. Perform standard ligation with 100 ng of vector and 100–1,000 ng of insert using 400 U T4 ligase at 14 °C for 16 h. Heat inactivate at 65 °C for 10 min.
  6. Transform competent bacteria using a standard heat-shock-protocol16.
  7. Plate on agar plates containing 15 µg/mL gentamicin. Incubate at 37 °C for 16 h.
  8. Pick single colonies, purify plasmid DNA, and verify insert by sequencing17 using the pCEP forward primer: 5' AGAGCTCGTTTAGTGAACCG 3'.
    NOTE: PCR on single colonies can be performed without prior purification with primers pCEP forward and pCEP reverse (5' AGA AAG CTG GGT CTA GAT ATC TCG 3'). This step can useful for pre-selection of positive colonies.
  9. Proceed to step 4.

3. Cloning of a miR-shRNA for Inducible Gene Knock-down

  1. Design miR-shRNA oligonucleotides according to the pSLIK cloning protocol19. Anneal and purify oligonucleotides and dilute 1 : 20 in ddH2O.
  2. Digest 3 µg of Entry vector with 5 U BfuAI at 50 °C for 3 h, then inactivate the reaction at 65 °C for 20 min.
    NOTE: If co-expression of green fluorescent protein (GFP) is desired, use pEN_TTGmiRc19 as an entry vector, otherwise use pEN_TTmiRc219.
  3. Gel purify digested vector as in step 2.5. Perform standard ligation of 100 ng of vector and 1 µL of purified and diluted shRNA oligonucleotides (step 3.1) using 400 U T4 ligase at room temperature for 1 h. Heat inactivate at 65 °C for 10 min.
  4. Transform competent bacteria using a standard heat-shock-protocol16.
  5. Plate on agar plates containing 15 µg/mL gentamicin. Incubate at 37 °C for 16 h.
  6. Pick single colonies, purify plasmid DNA, and verify insert by Sanger sequencing17 (sequencing primer: 5' TAGTCGACTAGGGATAACAG 3').
  7. Proceed to step 4.

4. Recombinational Cloning to Generate Ready-for-Injection Clones

  1. Mix 150 ng of Entry vector (from step 2 or step 3) and 150 ng of pTC TET-vector20.
  2. Add TE buffer to a total volume of 8 µL (pH=8).
  3. Transfer the LR-clonase enzyme mix II (see Table of Materials) to ice, incubate for 2 min. Vortex twice.
  4. Add 2 µL of LR-clonase enzyme mix II to the reaction and incubate at 25 °C for 1 h.
  5. Stop the reaction by adding 1 µL of Proteinase K-solution. Incubate at 37 °C for 10 min.
  6. Transform Stbl3 competent bacteria with 2 µL of recombinational cloning mix using a standard heat-shock-protocol16.
  7. Plate on agar plates containing 100 µg/mL ampicillin. Incubate at 30 °C for 24 h.
  8. Pick single colonies, purify plasmid DNA using an endotoxin-free plasmid preparation kit18 according to the manufacturer's instructions, and confirm vector integrity by Sanger sequencing17 (sequencing primer 5' AGGGACAGCAGAGATCCAGTTTGG 3').
  9. Construct is ready for injection, continue with step 5.

5. Preparing Solution for Hydrodynamic Tail Vein Injection

NOTE: Preparation of constructs for constitutive and inducible gene expression are described in Step 1, 2, 3, and 4.

  1. Prepare sterile 0.9% saline for injection (do not use PBS). Use volume corresponding to about 10% of mouse body weight. Example: for a mouse weighing 20 g, prepare 2 mL of solution.
  2. Prepare injection vectors that were purified using an endotoxin-free plasmid purification kit18 (see step 1.8 or 4.8, respectively).
  3. Add 10 µg or 15 µg of endotoxin-free sleeping beauty vector construct (from step 1 use 10 µg, from step 4 use 15 µg, respectively) and 1 µg of endotoxin-free pc-HSB521 per mL of sterile saline.
  4. Store solution for up to 4 h at 4 °C. Do not freeze.

6. Performing Hydrodynamic Tail Vein Injection

  1. Use a restrainer for tail vein injection (commercial or prepared from a 50 mL conical tube with holes for breathing and for the tail). Fill bottom of the tube with tissue paper.
  2. For injection, use mice of about 8–10 weeks of age with weights of 20–25 g.
  3. Weigh mice before injection and prepare injection volume according to body weight (corresponding to 10% of body weight, see step 5.1). Prepare a sterile 3 mL-syringe with a 27 G-needle for injection and fill with the required volume.
  4. Place the mouse into the restrainer. Adjust the amount of tissue paper (see step 6.1) to leave only minimal space for movement but enough space for breathing.
  5. Ensure that the mouse is breathing regularly.
  6. Warm the tail using an infrared lamp for 30-60 s. Carefully watch for signs of overheating.
  7. Clean the tail with an alcohol swab.
  8. Insert the needle almost horizontally into either one of the two lateral tail veins close to the base of the tail.
    NOTE: If placed successfully, a small amount of blood might flow back into the cone of the needle. It is not recommended to actively aspirate as any additional movement of the needle can result in its displacement and/or injury of the vein.
  9. Inject the total volume into the tail vein within 8-10 s.
  10. Immediately remove the mouse from the restrainer. Compress injection wound for at least 30 s or until any bleeding subsides.
  11. Place the mouse into a separate cage. Once the mouse has recovered from the procedure (about 30–60 min), transfer the mouse back to its original cage. Check on the mouse regularly for the next 24 h.
    NOTE: Mild sedation of the mouse is routinely observed for up to 2 h after injection.
  12. Before proceeding with further experiments (i.e., step 7), wait 10–15 days for clearance of non-integrated vectors.

7. Induction of Transfected CreER with Tamoxifen

CAUTION: Tamoxifen is harmful, may be cancerous or damage fertility. Please refer to the safety data sheet.

  1. Plan intraperitoneal tamoxifen injections on three consecutive days.
  2. On day 1, dissolve 10 mg of tamoxifen in 40 µL ethanol. Incubate at 55 °C for 10 min. Vortex several times until the tamoxifen has dissolved.
  3. Add 960 µL corn oil. Incubate for 5 mins at 55 °C. Vortex several times to get a clear solution.
  4. Prepare the solution in a 1-mL insulin syringe with a 27 G needle.
  5. Scruff the mouse by grabbing the neck of the mouse carefully with the thumb and the second finger, fixing the tail between the base of the hand and the fourth and fifth finger.
  6. Inject 0.1 mL (= 1 mg of tamoxifen) of the solution intraperitoneally into the left lower quadrant of the abdomen.
  7. Repeat the injections on days two and three.

8. Induction of Tetracycline-dependent Gene or shRNA Expression

CAUTION: Doxycycline may be harmful. Please refer to the safety data sheet.

NOTE: Depending on the type and duration of the experiment, doxycycline can be supplied in drinking water (step 8.1) or chow (step 8.2)

  1. For short term experiments (<10 days) administer doxycycline via drinking water using the following protocol.
    1. Dissolve 5 g sucrose in 100 mL of tap water. Autoclave.
    2. Dissolve 100 mg of doxycycline-hyclate in 5 mL of sucrose solution (step 8.1.1) in a 15-mL conical tube.
    3. Using a 10-mL syringe, sterile filter the solution through a 0.2 µm filter. Add to the sucrose solution prepared in step 8.1.1.
    4. Supply doxycycline-sucrose solution as drinking water to the mouse. Check daily and replace when the solution becomes cloudy indicating bacterial overgrowth (replace clear solution after three days at the latest).
  2. For long term experiments (>10 days) or experiments sensitive to metabolic changes, use commercial doxycycline-chow (e.g., Doxycycline Hyclate Chow 0.625 g/kg) to avoid dehydration and/or sucrose-induced changes in the livers of treated animals.

9. Preparation of Mouse Liver for Analysis by Immunostaining

CAUTION: Paraformaldehyde may be harmful. Please refer to the safety data sheet.

NOTE: The timepoint when mice will be analyzed depends on the experiment. It is recommended to analyze liver tissue after no less than three days of doxycycline treatment to ensure sufficient induction of transgene or shRNA expression.

  1. Prepare a 1 mL syringe with a 27 G needle with 1 mL of 4% paraformaldehyde solution (PFA).
  2. Euthanize the mouse by an appropriate method according to an approved animal protocol.
    NOTE: Guidelines for appropriate methods of euthanasia may vary depending on the institution.
  3. Using dissecting scissors and anatomical forceps, carefully open the abdominal cavity with a median laparotomy to expose the liver. Move the small intestine to the right to expose the portal vein and the inferior vena cava (IVC).
  4. Insert the needle of the prepared syringe (see step 9.1) into the IVC and cut the portal vein. Inject 1 mL of PFA slowly into the IVC to perfuse the liver tissue and remove auto-fluorescent red blood cells (desirable if immunofluorescent staining will be performed).
  5. Remove the liver. Rinse in water and transfer to 5–10 mL of 4% PFA solution.
  6. For paraffin sections, fix tissue for 36–48 h. Tissue is ready for dehydration and paraffin embedding.
  7. For frozen sections, fix tissue in 4% PFA for 1 h.
    1. For cryoprotection, transfer to 10% sucrose solution. Incubate 60 min.
    2. Transfer to 20% sucrose solution. Incubate 60 min.
    3. Transfer to 30% sucrose solution. Incubate 12–16 h. Embed in embedding compound for frozen sections and freeze at -20 °C.

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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 construct6,22,23. Additionally, the transfection efficiency is generally lower in larger...

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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 hepatocytes25,26. Predictors of a successful transfection are the injected volume per inj...

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Disclosures

The authors have nothing to disclose

Acknowledgements

This work was supported by Deutsche Krebshilfe, Germany (grant number 111289 to UE), the Lucile Packard Foundation for Children's Health (Ernest and Amelia Gallo Endowed Postdoctoral Fellowship - CTSA grant number UL1 RR025744 to UE). We thank Dr Mark A. Kay for vector constructs and experimental advice and Dr Julien Sage for mice and experimental support.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
General Material
GeneRuler 1 kb Plus DNA LadderThermo Fisher#SM1331DNA ladder for electrophoresis
Tissue-Tek O.C.T.Sakura4583embedding of cryo-sections
Biozym LE AgaroseBiozym840004
Ethidium bromideSigma-AldrichE7637-1G
D(+)-SaccharoseCarl Roth4621.1For sweetening of the doxycyline solution
Ampicillin Sodium SaltAppliChemA0839,0010For selection of Amp-resistant clones
LB Agar (Luria/Miller)Carl RothX969.1
LB Broth (Luria/Miller)Carl RothX968.1
S.O.C. MediumThermo Fischer15544034
Gentamicin sulfateAppliChemA1492,0001For selection of Gentamicin-resistant clones
Roti-Histofix 4 %Fa. RothP087.6para-formaldehyde solution
T4 DNA LigaseNew England BioLabsM0202S
GatewayTM LR ClonaseTM II Enzyme Mixinvitrogen/ThermoFisher11791-020contains LR-clonase enzyme mix II and proteinase K
DB3.1 Competent CellsThermo Fisher11782-018
Stbl3 Chemically Competent E. coliThermo FisherC737303
NameCompanyCatalog NumberComments
Restriction Enzymes
PacINew England BioLabsR0547S
AscINew England BioLabsR0558S
FseINew England BioLabsR0588S
SacINew England BioLabsR0156S
SpeINew England BioLabsR0133S
KpnINew England BioLabsR0142S
NotINew England BioLabsR0189S
XhoINew England BioLabsR0146S
BfuAINew England BioLabsR0701S
NameCompanyCatalog NumberComments
Kits
QIAquick Gel Extraction KitQiagen28704For DNA Extraction from gel
NucleoSpin Gel and PCR Clean UpMacherey & Nagel740609.10
NucleoBond PC20Macherey & Nagel740571Plasmid extraction (Mini prep)
NucleoBond PC500Macherey & Nagel740574Plasmid extraction (Maxi prep)
Phusion High-Fidelity DNA PolymeraseThermo FisherF530S
NameCompanyCatalog NumberComments
Materials for Mouse Experiments
Injekt Syringe F 1 mlBraun9166017VFor intraperitoneal injection
Omnifix Luer 3 mlBraun4616025VFor intravenous injection
Sterican Cannula 24GBraun4657675
Sterican Cannula 27GBraun4657705
TamoxifenSigma-AldrichT5648-1GFor CreER activation
Corn oilSigma-AldrichC8267-500MLCarrier for tamoxifen injections
Doxycycline hyclateAppliChemA2951,0025Activation of tetracycline-dependent expression
Injekt 10 ml SyringeBraun4606108V
Filtropur S 0.2Sarstedt831,826,001For filtration of doxycycline
NaCl 0,9%Braun3200905Carrier for intravenous injections
Falcon Conical Tube 50mlCorning Life Science352095
Infrared LampN/AN/AFor warming of mouse tail
IVISPerkin Elmer124262In vivo imaging system
NameCompanyCatalog NumberComments
Plasmids for cloning of sleeping beauty-transposon vectors for HTVI.
pTCn/aVector for constitutive gene expression, ref. 15
pEN_TTmcsAddgene #25755Entry vector for inducible gene expression, ref. 19
pEN_TTGmiRc2Addgene #25753Entry vector for inducible miR-shRNA expression with co-expression of GFP, ref. 19
pEN_TTmiRc2Addgene #25752Entry vector for inducible miR-shRNA expression without co-expression of GFP, ref. 19
pTC ApoE-TetAddgene #85578Expression vector for inducible gene or miR-shRNA expression with ApoE.HCR.hAAT promotor, ref. 11
pTC-CMV-TetAddgene #85577Expression vector for inducible gene or miR-shRNA expression with CMV promotor, ref. 11

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Sleeping Beauty TransposonCreER Inducible SystemGene Expression AnalysisLiver Tissue PreparationTamoxifen Induction ProtocolDoxycycline Administration MethodBioluminescence Imaging TechniquePlasmid Construct PreparationMouse Hepatocyte Transfection
Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection
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