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^2,3.

Classical techniques of generating GEMMs are often impaired by their complexity, timely expenses, and costs. Conditional knockout models have been widely used to study prostate cancer candidate genes, that result in embryonic lethality when inactivated in the germline⁴. Most common models involve a prostate-specific Cre recombinase driven by either a modified Probasin⁵ or a PSA⁶ promoter integrated in the GEMM by additional cross-breeding. In these models, the gene of interest will be targeted in the majority of prostate epithelial cells, generating hyperplasia in the entire organ, which may impair the animal's urinary tract function⁷.

Viral delivery of the Cre protein by injection into the anterior lobe of the murine prostate can resolve this problem by only targeting a few cells⁸. Taking laboratories technical prerequisites, expertise, and objectives into account, the method benefits from a broad range of possible variations. Successful approaches utilizing Adenovirus targeting JunB and Pten⁹ or Lentivirus targeting Pten and Trp53¹⁰ have been shown amongst others. Adding transgenes, such as luciferase, to the viral construct or to the GEMM will furthermore enable non-invasive monitoring of disease progression via bioluminescence imaging¹¹.

Genome editing based on the CRISPR/Cas9 technology reveals a new and rapid opportunity to study cancer through rapid generation of somatic knockouts¹². Viral delivery of single guide RNAs (sgRNAs) directed to the anterior lobe of the murine prostate establishes a physiologically more relevant model of prostate cancer. By this means, single cells carrying chosen mutations can form clones that are capable of expansion and invasion. Furthermore, use of guide RNAs for multiple target genes will generate cell clones with alterations in different genes. This will allow tumor heterogeneity and a natural selection pressure on cancer progression, which can reveal the importance of each gene alteration or epistatic mechanisms.

Here we present a method to deliver viral particles to the murine prostate for alteration of gene expression. By a small abdominal incision, the murine anterior prostate lobe is exposed and viral particles are injected into the lobe. Five days post-surgery, surgical clips can be removed from the skin and the prostatic cancer can be analyzed from 8 weeks after. Overall, this is a rapid and cost-efficient procedure, which has little impact on the mouse and allows larger tumor to develop without compromising the mouse.

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 injected at a titer of 1 x 10¹² IU/mL. The virus has serotype 9 and contains guide RNA to Pten and expression of Cre protein under a ubiquitin promotor (Figure 1). Loss of Pten increased pAKT and proliferation of the prostatic epithelium¹³.
2. Use male mice at an age of 8 weeks at the time of surgery to ensure adequate prostate development.
   NOTE: Mice shown in this experiment are Rosa26-LSL-Cas9-EGFP knockin mice on C57BL/6J background¹² (Figure 1).
3. Freshly prepare a general anesthesia mix and if possible keep it sterile.
   NOTE: For better animal recovery after surgery, an anesthesia antidote is highly recommended.
   1. To prepare a total volume of 5 mL anesthetic, mix 0.15 mL medetomidine hydrochloride (1 mg/mL), 0.4 mL midazolam (5 mg/mL), and 0.25 mL butorphanol (10 mg/mL) with 4.2 mL sterile saline water.
      NOTE: Alternative anesthesia methods, such as inhalable isoflurane, can be used according to the institutional animal care guidelines.
   2. To reverse the effect of medetomidine hydrochloride post-surgery, mix 0.1 mL atipamezole (5 mg/mL) with 4.9 mL sterile saline water.
4. Prepare an aseptic surgical environment by proper disinfection and sterilization. Autoclave surgical instruments before use.

2. Virus-delivery to the Murine Prostate

1. Anesthetize the animal by intraperitoneal injection with a sterile 1 mL syringe and a 27G x ½" needle. Use an anesthesia dose of 0.01 mL/g body weight. If the procedure lasts longer than 30 min, maintain anesthesia by injecting 1/3 of the starting dose every 30 min.
2. Examine the anesthetic depth by assessing muscle relaxation, pedal withdrawal, and palpebral reflexes. When loss of reflexes is observed, shave the lower abdomen of the animal.
3. Carefully cover the animal's eyes with veterinary ophthalmic ointment using a sterile cotton swab to prevent blindness due to lack of corneal reflex.
4. Wipe the shaved abdomen with 70% ethanol and 10% povidone iodine to disinfect the surgical area. Scrub the surgical area from the center of the surgical area toward the periphery. 
5. Perform a vertical skin incision at the low-abdominal midline of approximately 1 cm using a sterile surgical scissor.
6. Lift the peritoneum using a fine point forceps to prevent damaging the organs that are laying underneath. Carefully make an < 8 mm incision using surgical scissors through the peritoneum.
   NOTE: In contrast to human, a rodent's prostate gland comprises four separate lobes. The anterior lobe is attached to the seminal vesicle.
7. Gently move the fat tissue aside to uncover the seminal vesicle. Using a ring forceps, carefully lift up the seminal vesicle until the anterior prostate can be identified (Figure 1).
8. Inject a total volume of 30 µL virus (3 x 10¹⁰ viral particles) solution in the anterior prostate epithelium using a 0.5 mL insulin syringe with a 30G x 8 mm needle. Minimize leakage and ensure the fluid is absorbed within the tissue, forming a small bubble. Place the seminal vesicle back in the abdominal cavity.
9. Suture the peritoneum with 2-3 simple interrupted stitches using 6-0 absorbable sutures with a 13 mm, 3/8 circle, and a taper point needle.
10. Staple the skin with 3 sterile 4.8 x 6.5 mm clips lifting the skin with forceps to avoid damaging the peritoneum.
11. For better recovery, apply the anesthesia antidote in a dose of 0.01 mL/g body weight by intraperitoneal injection using a sterile 1 mL syringe and a 27G x ½" needle. Carefully place the animal back in its cage.

3. Post-surgery Procedures

1. Keep the cage with mice that underwent surgery on a heating pad for 1 h after the procedure, to prevent hypothermia until fully recovered. If the antidote is applied, the animal should be awake a few minutes after the injection.
2. Monitor the animals daily for appropriate wound healing and pain indications. If necessary, administer additional pain killers according to institutional animal care guidelines.
3. Remove skin clips after 4-5 days, once the wound is closed.

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