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Intraductal Injection of Lentivirus Vectors for Stably Introducing Genes into Rat Mammary Epithelial Cells in Vivo.

ABSTRACT: Various retroviral and lentiviral vectors have been used for up-the-teat intraductal injection to deliver markers, oncogenes, and other genes into mammary epithelial cells in mice. These methods along with the large number of genetically engineered mouse lines have greatly helped us learn normal breast development and tumorigenesis. Rats are also valuable models for studying human breast development and cancer. However, genetically engineered rats are still uncommon, and previous reports of intraductal injection of retroviral vectors into rats appear to be inefficient in generating mammary tumors. Here, we report, and describe the method for, stably introducing marker genes and oncogenes into mammary glands in rats using intraductal injection of commonly used lentiviral vectors. This method can infect mammary epithelial cells efficiently, and the infected cells can initiate tumorigenesis, including estrogen receptor-positive and hormone-dependent tumors, which are the most common subtype of human breast cancer but are yet still difficult to model in mice. This technique provides another tool for studying formation, prevention, and treatment of breast cancer, especially estrogen receptor-positive breast cancer.

SUBMITTER: Bu W

PROVIDER: S-EPMC7965254 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe aim of this study is to observe the inhibitive effects of p66Shc gene interfering lentivirus vectors on the expression of p66Shc, and to explore its effects on alveolar epithelial cells apoptosis induced by hyperoxia.MethodsThe gene sequences were cloned into the pLenR-GPH-shRNA lentiviral vector, which was selected by Genebank searches. The pLenR-GPH-shRNA and lentiviral vector packaging plasmid mix were cotransfected into 293T cells to package lentiviral particles. Culture virus supernatant was harvested, and then the virus titer was determined by serial dilution assay. A549 cells were transduced with the constructed lentiviral vectors, and real-time polymerase chain reaction (RT-PCR) and Western blot were used to evaluate p66Shc expression. This study is divided into a control group, a hyperoxia group, an A549-p66ShcshRNA hyperoxia group, and a negative lentivirus group. Cell apoptosis was detected by flow cytometry after 24 hours; the expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-9 were detected by immunohistochemistry assay. The production of reactive oxygen species and cellular mitochondria membrane potential (??m) were determined by fluorescence microscopy.ResultsWe successfully established the p66Shc gene interfering lentivirus vectors, A549-p66ShcshRNA. The A549-p66ShcshRNA was transfected into alveolar epithelial cells, and the inhibitive effects on the expression of p66Shc were observed. Both RT-PCR and Western blot demonstrated downregulation of p66Shc expression in A549 cells. In the A549-p66ShcshRNA hyperoxia group, we found dampened oxidative stress. A549-p66ShcshRNA can cause p66Shc gene silencing, reduce mitochondrial reactive oxygen species generation, reduce membrane potential decrease, reduce the apoptosis of A549 cells, and reduce alveolar epithelial cell injury, while the lentiviral empty vector group had no such changes.Conclusionp66Shc gene interfering lentivirus vector can affect the alveolar epithelial cells apoptosis induced by hyperoxia.

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Intraductal Injection of Lentivirus Vectors for Stably Introducing Genes into Rat Mammary Epithelial Cells in Vivo.

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