Mouse mammary tumor virus carrying a bacterial supF gene has wild-type pathogenicity and enables rapid isolation of...

Area of Science:

• Molecular Biology
• Virology
• Oncology

Background:

• Mouse mammary tumor virus (MMTV) is a common tool for insertional mutagenesis, identifying proto-oncogenes activated by viral insertions.
• Cloning MMTV insertion sites is crucial for understanding mammary carcinogenesis but can be labor-intensive.
• Existing methods require extensive screening to identify MMTV proviral integration sites.

Purpose of the Study:

• To develop a more efficient method for cloning MMTV insertion sites.
• To introduce a selectable marker into the MMTV genome to facilitate cloning.
• To validate the utility of the modified MMTV for rapid identification of proviral-cellular junctions.

Main Methods:

• A bacterial supF suppressor tRNA gene was inserted into the long terminal repeat (LTR) of MMTV, creating MMTVsupF.
• MMTVsupF was used to induce mammary tumors in mice, and the supF gene's stability was assessed.
• Proviral-cellular junction fragments from MMTVsupF-induced tumors were cloned into lambda vectors for selection on supF-deficient bacteria.

Main Results:

• The MMTVsupF virus induced mammary tumors and retained the supF gene stably through transmission.
• Cloning of MMTV insertion sites was significantly expedited by selecting for the supF gene.
• All tested lambda clones contained the desired proviral-cellular junction fragments, confirming the system's efficacy.

Conclusions:

• The MMTVsupF system provides a rapid and efficient method for cloning MMTV insertion sites.
• This approach circumvents traditional screening, accelerating the identification of MMTV-activated oncogenes.
• The modified MMTV is a valuable tool for molecular studies of insertional mutagenesis and cancer gene discovery.