BACKGROUND: Previous studies have demonstrated that LRP16 is an estrogen-responsive gene. Its expression level is strongly associated with the proliferation and invasive growth of human breast cancer cells.OBJECTIVE: To construct a LRP16 targeting vector and screen mouse embryonic stem cell clones with homolougous recombination of an inactive LRP16 gene.DESIGN: Constructing an inserting inactivation target by inserting SA-RIES-β geo expression cassette.SETTING: Bioregulatory Laboratory of the Third Medical Department of Kyushu University in Japan and Department of Molecular Biology, General Hospital of Chinese PLA.MATERIALS: The materials used here were mainly provided by the Bioregulatory Laboratory, the Third Medical Department of Kyushu University in Japan. The mouse genomic library in pBeloBAC11 Vector was purchased from lnvitrogen Corp. The competent TopF10 was purchased from Beijing Tiangen Biotech Corp. pcDNA3.1(+) vector was kept in our laboratory. Mouse ES cells were provided by Kyushu University.METHODS: The experiment was performed in Kyushu University and Department of Molecular Biology of PLA General Hospital from November 2004 to May 2005. Targeting sequence of LRP16 gene was obtained from 129 mouse genomic Bacterial Artificial Chromosomes library based on polymerase chain reaction (PCR) screening. The SA-RIES-β geo fragment was inserted within LRP16 fifth exon to inactivate LRP16. ES cells were screened with G418 and the homologously recombinant clone was identified by Southern blot analysis.MAIN OUTCOME MEASURES: Clones with homologous recombination.RESULTS: The LRP16 fragment including exon 5 to 11 was subcloned into the pBluescript SK Ⅱvector. Restriction map demonstrated that the SA-IRES-β geo fragment was correctly inserted into the LRP16 fifth exon. Southern blot results showed that there was an ES clone with targeting sequence homologously inserted.CONCLUSION: A LRP16 gene targeting vector is constructed and a homologous recombinant is obtained.

A 68-year-old woman received aortic valve replacement (AVR) with a Freestyle stentless valve using a subcoronary technique for aortic stenosis and regurgitation in September 2000. She complained of chest pain, had low grade fever and findings of inflammation and was admitted to our hospital with a diagnosis of acute myocardial infarction in December 2000. She suffered from repetitive or recurrent myocardial infarction. Transesophageal echocardiogram revealed no abnormal findings of the Freestyle stentless valve, but her blood culture was positive for methicillin-resistant coagulase negative Staphylococcus aureus (MRCNS) and she underwent an emergency operation. The Freestyle stentless valve was removed and replaced with a mechanical valve. The patient's intraoperative tissue grew MRCNS and parenteral antibiotics were administered for 8 weeks after surgery. Her condition was complicated with multiple cerebral infarction, however she was discharged on the 113th postoperative day and is doing well without recurrence of infection 12 months after the operation.