Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

Summary

This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

Abstract

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease.

Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane.

This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

Introduction

Detection and treatment of prostate cancer have significantly improved over the last decade. Still, the incidence of prostate cancer is increasing, following life expectancy. With an estimated 1.1 million new cases worldwide, it is among the most common causes of cancer-related death in men ¹. Prostate cancer is slow in its development, but when the cancer has progressed to an advanced metastatic state, prognosis is poor due to limited treatment options. So far, only a few genes have been identified as common drivers in this cancer, and its heterogeneity and multifocality impedes detection of biomarkers and targetable disease drivers

Protocol

This protocol involves a surgical procedure in laboratory mice. All animal experiments must be individually reviewed and approved by an Institutional Animal Care and Use Committee (IACUC). As the approach is based on animal recovery and survival, ensure appropriate anesthesia, pain management, and an aseptic surgical environment at all time. Use a heating pad to prevent hypothermia during surgery and until recovery from anesthesia.

1. Starting Considerations

1. Depending on the virus used, different titer requirements may apply; dilute the virus accordingly.
   NOTE: In this example, an Adeno-associated virus diluted in PBS is ....

Results

To assess virus delivery to the murine prostate, samples were analyzed three months after the surgery. The Rosa26-LSL-Cas9-EGFP mice¹² express GFP in cells that have been exposed to Cre protein expressed by the virus. The prostate samples were examined with a fluorescence microscopy to identify areas with GFP signal (Figure 2A). The GFP signal indicates Cre activity in the prostate epithelium but not whether gene editing has been induc.......

Discussion

In this protocol, we describe a method to alter gene expression in the anterior lobe of the murine prostate by virus injection, creating a powerful new mouse model for prostate cancer (Figure 2). The successful administration of an Adenovirus was first described by Leow et al. in 2005⁸. We have previously shown how an Adenovirus coding for a Cre recombinase protein can replace time-consuming cross-breeding of a Cre allele for tissue-specific deletion

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
B6J.129(B6N)-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J mice	The Jackson Laboratory	26175
0,5 ml U-100 insulin syringe	BD	324825	for virus injection
1 ml syringe	BD	300013	for anesthesia injection
30 G 1/2'' needle	BD	304000	for anesthesia injection
6-0 Polysorb Suture	Medtronic	GL889	to close the peritoneum
Disposable sterilized surgery drape	multiple suppliers
Heating pad	multiple suppliers
Povidone-Iodine Prep Pad	Fisher Scientific	06-669-70
Trimming machine	Aesculap	GT415	to shave the abdomen
Dumont Forceps	F.S.T	11252-00
Halsey Micro Needle Holder	F.S.T	12500-12
Iris Scissors	F.S.T	14094-11
Narrow Pattern Forceps	F.S.T	11002-12
Ring Forceps	F.S.T	11106-09
Wound Clip System Handle incl. Clips	F.S.T	12030-01	to close the skin
Wound Clip System Remover	F.S.T	12030-04
Microscope	Leica		different models have been used
Reagents
1x PBS	Gibco	10010-023
Antisedan			obtained from the animal facility
Butorphanol			obtained from the animal facility
Medetomidinhydrochlorid			obtained from the animal facility
Midazolam			obtained from the animal facility
100% Ethanol	Fisher Scientific	22-032-103
Eye ointment	Takeda	7242
Virus (AAV, AV)	multiple suppliers		virus used in this protocol is an in-house production
pAKT antibody	CST	4060	used 1:200 dillution
GFP anitbody	CST	2956	used 1:100 dillution