MR was funded by a fellowship from the Danish cancer society (R146-A9394-16-S2). MFB and MKT were funded by AUFF NOVA (AUFF-E-2015-FLS-9-8). MR and MFB were co-funded by Graduate School, HEALTH, AU. The E.F.W. laboratory is supported by grants from the Spanish Ministry of Economy (SAF2015-70857, co-funded by the ERDF-EU) and an ERC advanced grant (741888 - CSI-Fun).
We want to thank Liliana Fajardo Mellor (Genes, Development and Disease; National Cancer Research Center) for critical reading of the manuscript.

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
<ol>
	<li>Depending on the virus ...

<ol>
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A., De Gasperi, R., Elder, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+transgenesis:+an+overview.">Animal transgenesis: an overview.</a> Brain Struct Funct. 214, 91-109 (2010).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+prostate+epithelial+cell-specific+Cre+transgenic+mouse+model+for+tissue-specific+gene+ablation.">Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation.</a> Mech Dev. 101, 61-69 (2001).</li><li>Ma, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+biallelic+inactivation+of+Pten+in+the+mouse+prostate+leads+to+prostate+cancer+accompanied+by+increased+epithelial+cell+proliferation+but+not+by+reduced+apoptosis.">Targeted biallelic inactivation of Pten in the mouse prostate leads to prostate cancer accompanied by increased epithelial cell proliferation but not by reduced apoptosis.</a> Cancer Res. 65, 5730-5739 (2005).</li><li>Chen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+role+of+p53-dependent+cellular+senescence+in+suppression+of+Pten-deficient+tumorigenesis.">Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis.</a> Nature. 436, 725-730 (2005).</li><li>Leow, C. C., Wang, X. D., Gao, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+method+of+generating+prostate-specific+Cre-LoxP+gene+switching+via+intraductal+delivery+of+adenovirus.">Novel method of generating prostate-specific Cre-LoxP gene switching via intraductal delivery of adenovirus.</a> Prostate. 65, 1-9 (2005).</li><li>Thomsen, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+JUNB/AP-1+promotes+invasive+prostate+cancer.">Loss of JUNB/AP-1 promotes invasive prostate cancer.</a> Cell Death Differ. 22, 574-582 (2015).</li><li>Cho, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+novel+GEM+model+for+metastatic+prostate+cancer+analysis+and+therapy,+reveals+myc+as+a+driver+of+Pten-mutant+metastasis.">a novel GEM model for metastatic prostate cancer analysis and therapy, reveals myc as a driver of Pten-mutant metastasis.</a> Cancer Discov. 4, 318-333 (2014).</li><li>Cho, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+in+vivo+validation+of+candidate+drivers+derived+from+the+PTEN-mutant+prostate+metastasis+genome.">Rapid in vivo validation of candidate drivers derived from the PTEN-mutant prostate metastasis genome.</a> Methods. 77-78, 197-204 (2015).</li><li>Platt, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas9+knockin+mice+for+genome+editing+and+cancer+modeling.">CRISPR-Cas9 knockin mice for genome editing and cancer modeling.</a> Cell. 159, 440-455 (2014).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostate-specific+deletion+of+the+murine+Pten+tumor+suppressor+gene+leads+to+metastatic+prostate+cancer.">Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer.</a> Cancer Cell. 4, 209-221 (2003).</li><li>Liu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copy+number+analysis+indicates+monoclonal+origin+of+lethal+metastatic+prostate+cancer.">Copy number analysis indicates monoclonal origin of lethal metastatic prostate cancer.</a> Nat Med. 15, 559-565 (2009).</li><li>Haffner, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+clonal+origin+of+lethal+prostate+cancer.">Tracking the clonal origin of lethal prostate cancer.</a> J Clin Invest. 123, 4918-4922 (2013).</li><li>Daya, S., Berns, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+using+adeno-associated+virus+vectors.">Gene therapy using adeno-associated virus vectors.</a> Clin Microbiol Rev. 21, 583-593 (2008).</li><li>Price, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Aspects+of+Development+and+Structure+in+the+Prostate.">Comparative Aspects of Development and Structure in the Prostate.</a> Natl Cancer Inst Monogr. 12, 1-27 (1963).</li><li>McNeal, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+prostate:+an+historical+survey+of+divergent+views.">Anatomy of the prostate: an historical survey of divergent views.</a> Prostate. 1, 3-13 (1980).</li><li>Thomsen, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX9+elevation+in+the+prostate+promotes+proliferation+and+cooperates+with+PTEN+loss+to+drive+tumor+formation.">SOX9 elevation in the prostate promotes proliferation and cooperates with PTEN loss to drive tumor formation.</a> Cancer Res. 70, 979-987 (2010).</li></ol>

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 <sup cla...

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 drivers2,3.
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&#39;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.

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B6J.129(B6N)-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J mice</td>
			<td>The Jackson Laboratory</td>
			<td>26175</td>
			<td></td>
		</tr>
		<tr>
			<td>0,5 ml U-100 insulin syringe</td>
			<td>BD</td>
			<td>324825</td>
			<td>for virus injection</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD</td>
			<td>300013</td>
			<td>for anesthesia injection</td>
		</tr>
		<tr>
			<td>30 G 1/2&#39;&#39; needle</td>
			<td>BD</td>
			<td>304000</td>
			<td>for anesthesia injection</td>
		</tr>
		<tr>
			<td>6-0 Polysorb Suture</td>
			<td>Medtronic</td>
			<td>GL889</td>
			<td>to close the peritoneum</td>
		</tr>
		<tr>
			<td>Disposable sterilized surgery drape</td>
			<td>multiple suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>multiple suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine Prep Pad</td>
			<td>Fisher Scientific</td>
			<td>06-669-70</td>
			<td></td>
		</tr>
		<tr>
			<td>Trimming machine</td>
			<td>Aesculap</td>
			<td>GT415</td>
			<td>to shave the abdomen</td>
		</tr>
		<tr>
			<td>Dumont Forceps</td>
			<td>F.S.T</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey Micro Needle Holder</td>
			<td>F.S.T</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>F.S.T</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow Pattern Forceps</td>
			<td>F.S.T</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring Forceps</td>
			<td>F.S.T</td>
			<td>11106-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound Clip System Handle incl. Clips</td>
			<td>F.S.T</td>
			<td>12030-01</td>
			<td>to close the skin</td>
		</tr>
		<tr>
			<td>Wound Clip System Remover</td>
			<td>F.S.T</td>
			<td>12030-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>different models have been used</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Antisedan</td>
			<td></td>
			<td></td>
			<td>obtained from the animal facility</td>
		</tr>
		<tr>
			<td>Butorphanol</td>
			<td></td>
			<td></td>
			<td>obtained from the animal facility</td>
		</tr>
		<tr>
			<td>Medetomidinhydrochlorid</td>
			<td></td>
			<td></td>
			<td>obtained from the animal facility</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td></td>
			<td></td>
			<td>obtained from the animal facility</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Fisher Scientific</td>
			<td>22-032-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Takeda</td>
			<td>7242</td>
			<td></td>
		</tr>
		<tr>
			<td>Virus (AAV, AV)</td>
			<td>multiple suppliers</td>
			<td></td>
			<td>virus used in this protocol is an in-house production</td>
		</tr>
		<tr>
			<td>pAKT antibody</td>
			<td>CST</td>
			<td>4060</td>
			<td>used 1:200 dillution</td>
		</tr>
		<tr>
			<td>GFP anitbody</td>
			<td>CST</td>
			<td>2956</td>
			<td>used 1:100 dillution</td>
		</tr>
	</tbody>
</table>

virus delivery of crispr guides to the murine prostate for gene alteration

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.

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...

Watch this Scientific Journal Video about Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration at JoVE.com

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

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.

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

The overall goal of this surgical procedure is to alter gene expression in the murine prostate by viral delivery of CRISPR Cas9 guides for prostate cancer induction. This method can help addressing new questions in prostate cancer biology regards to analyzing and validating new genes in an in vivo setting. The main advantages of this technique is it allow rapid gene editing of multiple genes by only targeting a subset of the prostatic cells as seen in the human scenario.Someone who is new to the procedure may struggle at first because of organ size, and the precision that is needed for a successful surgical completion. To freshly prepare a total volume of 5 milliliters of anesthetic, mix 0.15 milliliters of one mg per milliliter medetomidine hydrochloride, 0.4 milliliters of 5 mg per milliliter of midazolam, and 0.25 milliliters of 10 mg per milliliter butorphenol. Then, add 4.2 milliliters of sterile saline water.To reverse the effects of medetomidine hydrochloride post surgery, mix 0.1 milliliter of 5 mg per milliliter atipamezole with 4.9 milliliters of sterile saline water as an anesthesia antidote. Prepare an aseptic surgical environment by proper disinfection and sterilization and autoclave the surgical instruments. To deliver virus to the prostate, after anesthetizing an eight-week-old male mouse according to the text protocol;examine the anesthetic depth by assessing muscle relaxation, pedal withdrawal, and palpable reflexes.When loss of reflexes is observed, shave the lower abdomen of the animal. Using a sterile cotton swab, carefully cover the animal's eyes with veterinary ophthalmic ointment to prevent blindness caused by xerophthalmia. Then, use 70%ethanol and 10%providone iodine to wipe the shaved abdomen to disinfect the surgical area.Next, using sterile surgical scissors, make an approximately one centimeter vertical skin incision at the low abdominal midline. Then, with a fine point forceps, lift the peritoneum to prevent damaging the organs that are laying underneath;and use surgical scissors to carefully make an eight millimeter or shorter incision through the peritoneum. Gently move the fat tissue aside to uncover the seminal vesicle.Then, using a ring forceps, carefully lift up the seminal vesicle until the anterior prostate can be identified. Now, with a 0.5 milliliter insulin syringe and a 30 gauge 8 millimeter needle;inject a total volume of 30 microliters of virus solution into the anterior prostate epithelium. Minimize leakage and ensure the fluid is absorbed within the tissue, forming a small bubble.Then, place the seminal vesicle back in the abdominal cavity. To ensure the fluid is absorbed within the tissue in a small bubble and without leakage, make sure to inject parallel to the seminal vesicle and following the shape of the anterior prostate lobe. With a taper point needle and a 13 millimeter 3/8 circle, suture the perineum with two to three simple interrupted stitches of 6-0 absorbable suture.Then, lifting the skin with forceps to avoid damaging the peritoneum;staple the skin with three sterile 4.8 by a 6.5 millimeter clips. For better recovery following surgery, use a sterile 1 milliliter syringe and a 27 gauge by 1/2 inch needle to administer the anesthesia antidote in a dose of 0.1 milliliters per gram body weight by intraperitoneal injection. Then, carefully place the animal back in its cage.Keep the cage on a heating pad for one hour after the procedure to prevent hypothermia until fully recovered. The animal should awaken a few minutes after the injection. Continue to monitor and treat the animal daily according to the text protocol.And, when the wound has closed, remove the skin clips. To assess virus delivery to the murine prostate, samples were analyzed three months after surgery. As shown here, the mice used in this study expressed GFP in cells that have been exposed to the Cree protein expressed by the virus.The GFP signal shows Cree activity in the prostate epithelium but not whether gene editing had been induced by the CRISPR guide. These immunohistochemical sections identify focal areas of cells with high expressions of pAKT, indicating loss of P10. In these panels, costaining for pAKT and GFP identified double positive cells, which confirmed that transformation of prostatic cells by the adeno-associated virus.Over all, these results show that in vivo, CRISPR-Cas9 gene editing can be performed in the prostate epithelium by using the Rosa26-LSL-Cas9-EGFP mouse and the adeno-associated virus. Once mastered, the procedure can be done in 15 minutes. This technique paved the way for research in the field of prostate cancer to examine multiple genes with simultaneous in vivo.After watching this video, you should have a good understanding of how to deliver substrate to the murine prostate. Don't forget that when working with some viruses can be extremely hazardous and precautions should always be taken when doing these procedures.

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8.5K visualizações.  Aarhus University. The overall goal of this surgical procedure is to alter gene expression in the murine prostate by viral delivery of CRISPR Cas9 guides for prostate cancer induction. This method can help addressing new questions in prostate cancer biology regards to analyzing and validating new genes in an in vivo setting. The main advantages of this technique is it allow rapid gene editing of multiple genes by only targeting a subset of the prostatic cells as seen in the human scenario.Someone who is ...