Name: modeling breast cancer via an intraductal injection of cre-expressing adenovirus into the mouse mammary gland
Uploaded: 2019-06-07T13:30:00
Description: Watch this Scientific Journal Video about Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland at JoVE.com

This work was supported by National Institutes of Health (NIH) grant R01 CA222560 and by Department of Defense Breakthrough Award W81XWH-18-1-0037.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Brigham and Women&#8217;s Hospital.
1. Generation and maintenance of floxed mice
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	<li>Obtain breast cancer-relevant floxed conditional knockout (e.g., Trp53tm1Brn [referred to as Trp53L/L], Brca1tm1Aash [Brca1L/L]) or conditional knock-in mouse lines (e.g., Gt(ROSA)26Sortm1(Pik3ca*H1047R)Egan<...

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The success of this approach for inducing mammary tumors from different subpopulations of MECs relies not only on choosing appropriate cell-type-specific promoters (to drive Cre expression) but also on the intraductal injection procedure itself. The idea behind this approach is that the injected Ad-Cre viruses are retained in the ductal tree, which is a concealed structure with lumen, and therefore, only MECs are exposed to the viruses and are infected by Ad-Cre. Due to the limited lumen space within the mammary ducts, i...

The overall goal of this method is to develop a new breast cancer modeling approach based on an intraductal injection of Ad-Cre into the mouse MG. The Cre/loxP recombination-based genetic approach has been widely used to model human breast cancer in mice. The first generation of Cre/loxP-based breast cancer mouse models are generated by using Cre-expressing transgenic mice under the control of MEC-specific promoters (e.g., MMTV-Cre for luminal MECs and a portion of basal MECs, Wap-Cre and Blg-Cre for luminal progenitors and alveolar luminal MECs, K14-Cre for basal and a portion of luminal MECs1,2,3,4,5)6,7,8,9. However, while these Cre transgenic lines enable spatial control of Cre expression (i.e., in different subsets of MECs), they do not allow temporal control of Cre expression and Cre/loxP-mediated genetic manipulation. The second generation of Cre/loxP-based breast cancer mouse models utilize inducible Cre activity/expression approaches (e.g., use of Cre-estrogen receptor fusion [CreER], which can only induce Cre/loxP recombination upon administration of tamoxifen), and as a result, these genetic tools permit both spatial and temporal controls of the activation of oncogenic events in MECs (e.g., K8-CreER- and K5-CreER-based models)10,11,12. In both generations of breast cancer mouse models, as promoters used to drive Cre or CreER expression (e.g., Krt8, Krt5) may also be active in epithelial cells of other organs (i.e., they are cell-type-specific but not organ-specific) or have a leaky expression in cell types other than epithelial cells (e.g., MMTV, which has leaky activity in bone marrow hematopoietic cells), these approaches may lead to the development of unwanted cancer(s) in other organ(s). If these unexpected cancers cause lethality in the affected mice, the original purpose of modeling breast cancer in these mice may be prohibited (e.g., MMTV-Cre-driven oncogenic events may lead to hematopoietic malignancies and early death of the mice, due to leakiness of the MMTV promoter in hematopoietic cells)4.
Here we report a breast cancer modeling approach in mice that allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner. This approach is based on an intraductal injection of Ad-Cre into mouse MGs (and is, thus, organ-specific). Cre expression can be controlled by using different MEC subpopulation-specific promoters embedded in the adenoviral vector (e.g., Krt8 for luminal MECs, Krt5 for basal MECs, thus achieving cell-type specificity). Cancer induction in MGs can be temporally controlled by an injection of Ad-Cre into mice at different ages, starting from 3-4 weeks of age (pubertal) to the adult stage.

<table>
	<tbody>
		<tr>
			<td>33-gauge needle</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td>point style 3 blunt</td>
		</tr>
		<tr>
			<td>7mm Reflex Clip</td>
			<td>Braintree Scientific</td>
			<td>RF7 CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenovirus, Ad-K5-Cre</td>
			<td>University of Iowa Viral Vector Core</td>
			<td>Ad5-bk5-Cre (VVC-Berns-1547)</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenovirus, Ad-K8-Cre</td>
			<td>University of Iowa Viral Vector Core</td>
			<td>Ad5mK8-nlsCre</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td>Fisher</td>
			<td>HC800-1GAL</td>
			<td>Prepare to 70% in use</td>
		</tr>
		<tr>
			<td>biotinylated CD31</td>
			<td>eBiosciences</td>
			<td>13-0311-85</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated CD45</td>
			<td>eBiosciences</td>
			<td>13-0451-85</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated TER119</td>
			<td>eBiosciences</td>
			<td>13-5921-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>CD24-AF-700</td>
			<td>BD Pharmingen</td>
			<td>564237</td>
			<td></td>
		</tr>
		<tr>
			<td>CD24-PE</td>
			<td>eBiosciences</td>
			<td>12-0242-83</td>
			<td></td>
		</tr>
		<tr>
			<td>CD29-APC</td>
			<td>eBiosciences</td>
			<td>17-0291-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD29-PE</td>
			<td>eBiosciences</td>
			<td>12-0291-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair Remover Lotion</td>
			<td>Nair</td>
			<td></td>
			<td>9 Oz</td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>7636-01</td>
			<td>0.025 mL</td>
		</tr>
		<tr>
			<td>Iodophors</td>
			<td>Betadine</td>
			<td></td>
			<td>10% Povidone-iodine</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>NDC 10019-360-40</td>
			<td>1-2.5%</td>
		</tr>
		<tr>
			<td>Loxicam</td>
			<td>Norbrook</td>
			<td>NDC 55529-040-10</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>Lubricant Eye Ointment</td>
			<td>Akorn</td>
			<td colspan="2">NDC 17478-062-35</td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Pentair</td>
			<td>9M</td>
			<td>Watchmaker&#39;s Forceps</td>
		</tr>
		<tr>
			<td>Micro-dissecting tweezers</td>
			<td>Dumont</td>
			<td>M5</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq 5X Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0285L</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling breast cancer via an intraductal injection of cre-expressing adenovirus into the mouse mammary gland

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.

Representative PCR genotyping results for the R26Y and Trp53L alleles are shown in Figure 1.
Although, in principle, all 10 MGs can be subjected to the intraductal injection procedure, practically, the two fourth inguinal MGs are typically selected for injection, due to their easier accessibility and larger MG sizes (Figure 2). During the su...

Watch this Scientific Journal Video about Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland at JoVE.com

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

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