Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines

Lentiviral vectors (LVs) are considered one of the most promising vehicles to efficiently deliver genetic information for basic research and gene therapy approaches. Combining LVs with drug-inducible expression systems should allow tight control of transgene expression with minimal side effect on relevant target cells. A new doxycycline-regulated system based on the original TetR repressor was developed in 1998 as an alternative to the TetR-VP16 chimeras (tTA and rtTA) to avoid secondary effects due to the expression of transactivator domains. However, previously described TetR-based systems required cell cloning and/or antibiotic selection of tetracycline-responsive cells in order to achieve good regulation. In the present manuscript we have constructed a dual Tet-ON system based on two lentiviral vectors, one expressing the TetR through the spleen focus forming virus (SFFV) promoter (STetR) and a second expressing eGFP through the regulatable CMV-TetO promoter (CTetOE). Using these vectors we have demonstrated that the TetR repressor, contrary to the reverse transactivator (rtTA), can be expressed in excess to bind and modulate a high number of TetO operons. We have also showed that this dual vector system can generate regulatable bulk cell lines (expressing high levels of TetR) that are able to modulate transgene expression either by varying doxycycline concentration and/or by varying the amount of CTetOE vector genomes per cell. Based on these results we have developed a new all-in-one lentiviral vector (CEST) driving the expression of TetR through the SFFV promoter and the expression of eGFP through the doxycycline-responsive CMV-TetO operon. This vector efficiently produced Tet-ON regulatable immortalized (293T) and primary (human mesenchymal stem cells and human primary fibroblasts) cells. Bulk doxycycline-responsive cell lines express high levels of the transgene with low amount of doxycycline and are phenotypically indistinct from its parental cells

Hormone replacement therapy and cataract: a population-based case-control study.

PURPOSE: Laboratory studies have suggested that hormone replacement therapy (HRT) may protect against the development of cataract, but epidemiological studies in humans have thus far been inconclusive. The aim of this study was to assess the association between hormone replacement therapy and cataract. METHODS: Population-based case-control study using data from the General Practice Research Database in the UK. Participants were 10 000 women aged 45 years and over with diagnosed cataract and 10 000 controls matched on age, general practice, and calendar period. RESULTS: The crude odds ratio for the association between cataract and ever-use of oestrogen-only hormone replacement therapy was 1.13 (95% CI 0.99-1.29). This reduced to 0.81 (95% CI 0.71-0.94) after adjustment for consultation rate. Similarly, the crude odds ratio for the association between cataract and ever-use of a formulation containing oestrogen and progestogen was 1.18 (95% CI 1.01-1.39), reducing to 0.86 (95% CI 0.72-1.02) after adjustment for consultation rate. CONCLUSIONS: Oestrogen-only and oestrogen-progestogen hormone replacement therapies are associated with a small reduced risk of cataract. This data adds to the growing body of evidence on the effects of HRT on health. All potential benefits and risks of this therapy should be taken into account when considering its use