Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase - PubMed (original) (raw)

Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase

Michel DuPage et al. Nat Protoc. 2009.

Abstract

The development of animal models of lung cancer is critical to our understanding and treatment of the human disease. Conditional mouse models provide new opportunities for testing novel chemopreventatives, therapeutics and screening methods that are not possible with cultured cell lines or xenograft models. This protocol describes how to initiate tumors in two conditional genetic models of human non-small cell lung cancer (NSCLC) using the activation of oncogenic K-ras alone or in combination with the loss of function of p53. We discuss methods for sporadic expression of Cre in the lungs through engineered adenovirus or lentivirus, and provide a detailed protocol for the administration of the virus by intranasal inhalation or intratracheal intubation. The protocol requires 1-5 min per mouse with an additional 30-45 min to set-up and allow for the recovery of mice from anesthesia. Mice may be analyzed for tumor formation and progression starting 2-3 weeks after infection.

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Figures

Figure 1

Figure 1. Intratracheal infection technique

Anesthetized mice are placed on the platform by their front teeth so that their chest hangs vertically beneath them (a, b). The light is directed on the mouse’s upper chest (a, c), on the spot marked by the ‘X’ (c). The mouth is opened using the Exel Safelet IV catheter (d), and the tongue is gently pulled out using the flat forceps. After locating the white light emitted from the trachea (e), the Exel Safelet IV catheter is slid into the trachea (f), and the needle is removed (g). The mouse with the inserted catheter (h) on the platform is moved into a biosafety hood, where the virus is dispensed into the opening of the catheter (i). CAUTION All experiments should be done in accordance with protocols approved by the Institutional Animal Care and Use Committee.

Figure 2

Figure 2. Intranasal infection technique

The anesthetized mouse lies gently in the hand of the investigator (a), and the virus is administered dropwise (b) into one nostril until the virus is completely inhaled (b, c). CAUTION All experiments should be done in accordance with protocols approved by the Institutional Animal Care and Use Committee.

Figure 3

Figure 3. Survival is reduced in KP model compared to K model

Kaplan-Meier plot of KP mice (median survival 76 days) and K mice (median survival 185 days) infected with 2.5×107 PFU of Ad-Cre per mouse (provided by T. Oliver) (a). Kaplan-Meier plot of KP mice (median survival 170 days) and K mice (median survival 266 days) infected with 105 Lenti-Cre viruses per mouse (provided by P. Sandy and M. DuPage) (b). CAUTION All experiments should be done in accordance with protocols approved by the Institutional Animal Care and Use Committee.

Figure 4

Figure 4. Tumor progression and histopathological phenotype in KP model

Haematoxylin and eosin stained (H&E) tumors from KP mice at various times after infection with Lenti-Cre: (a) Grade 1 lesion of an AAH progressing to a small adenoma. (b) Grade 2 adenoma. (c) Uniform nuclei in a grade 2 adenoma. (d) Pleomorphic nuclei in a Grade 3 adenocarcinoma. (e) Grade 3 adenocarcinoma displaying mixed cellular phenotypes. (f) Grade 4 invasive adenocarcinoma. (g) Grade 4 adenocarcinoma with glandular/acinar architecture and desmoplasia. (h) Adenocarcinoma from the lung (L) invading across the mesothelium into the pleural cavity (P). (i) Local lung tumor metastasis (T) to mediastinal lymph node (LN). (j) Distant lung tumor metastasis (T) to the kidney (K). Panels a, b, e, f, i and j are at 100X magnification. Panels c, d, g and h are at 400X magnification. Scale bar = 100 µm. CAUTION All experiments should be done in accordance with protocols approved by the Institutional Animal Care and Use Committee.

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