Efficient Gene Knockdown in the Liver via Intrasplenic Injection of Adeno-Associated Virus Serotype 8 (AAV8)-Delivered Small Hairpin RNA

Summary

This protocol describes how intrasplenic injection of AAV8-delivered small hairpin RNA achieves the same gene knockdown efficiency in the liver as portal vein injection, representing a simpler procedure with much lower perioperative and postoperative mortality and complications.

Abstract

The liver is a major organ that performs essential metabolic functions. Developing an efficient and safe method to knock down gene expression in the liver provides an important tool for determining gene function in liver pathophysiology. In this study, we describe a method for intrasplenic injection of adeno-associated virus serotype 8 (AAV8) engineered to express a small hairpin RNA (shRNA) against a target gene of interest, nucleostemin (NS). Intrasplenic injection of AAV8 expressing an NS-targeting shRNA (AAV8-shNS1) achieved the same knockdown efficiency of NS in the liver as did portal vein injection, compared to the injection of AAV8 expressing a scrambled sequence shRNA (AAV8-shScr). Furthermore, injection of the AAV8-shRNA virus triggered minimal inflammatory reactions in the liver parenchyma. Most importantly, this intrasplenic injection protocol was not technically demanding and caused minimal bleeding at the injection site, which is the leading cause of perioperative and postoperative mortality when performing portal vein injection. This study reports an improved and relatively safe method to achieve efficient gene knockdown in the liver.

Introduction

The liver is a vital organ that metabolizes nutrients and chemicals, but is also under constant exposure to cytotoxic and carcinogenic insults. While the adult liver is capable of regrowing after injury, its regenerative power is severely hampered by age¹. To date, the only therapeutic option for patients with end-stage chronic liver diseases or massive acute liver damage is liver transplantation, which poses many challenges of its own². To better understand the molecular pathophysiology of the liver by interrogating the functions of genes of interest, genetic manipulations have been developed for in vivo a....

Protocol

All animal experiments completed in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the Texas A&M University Health Science Center in Houston (approval number: 2021-0264-IBT) and performed in accordance and compliance with all relevant regulatory and institutional guidelines. Female C57BL/6J mice (4-5 months old, body weight 23-30 g) were used. The details of the reagents and equipment used are listed in the Table of Materials.

Discussion

Gene KD by viral delivery of either a shRNA-expressing construct in a wildtype background^24,25 or Cre recombinase-expressing construct in a floxed background²⁶ is a powerful way to interrogate gene function in vivo in an inducible, time-controlled manner. An ideal delivery method for in vivo gene KO/KD studies should achieve a high KO/KD efficiency in the organ of interest, and, in addition, the procedure itself should be.......

Materials

Name	Company	Catalog Number	Comments
Amicon filter centrifugation units	MilliporeSigma	UFC9100	Fast ultrafiltration
AV5-siRNA-GFP	Addgene	#124972	Plasmid
Competent DH5α bacteria	Invitrogen	#18265-017	Chemically competent strain for cloning
HpaI	New England Biolabs	R0105	Restriction enzyme
iMFectin transfection reagent	GenDepot	I7100-101
M-MLV reverse transcriptase	Promega	M1708	RNA-dependent DNA polymerase
MyiQ single-color real-time PCR detection system	Bio-Rad	BUN9740RAD	qRT-PCR
Omega endotoxin-free kit	Bioteck	D6915-03	Plasmid DNA midi prep
Random hexamers	Invitrogen	48190-011
Supermix SYBR green reagent	Bio-Rad	1708882	Real-time PCR applications
T4 DNA Ligase	Promega	M1801
TRIzol Reagent	Life Technologies	15596-018	Isolation of high-quality total RNA
XhoI	New England Biolabs	R0146	Restriction enzyme