Novel dynamic culture system to support initiation of primordial follicle growth in prepubertal mouse ovaries.

OBJECTIVE: To evaluate the impact of dynamic in vitro culture on initiation of early follicular growth in prepubertal mouse ovaries. DESIGN: Ovaries from 8-day-old BALB/c mice were cultured either in a dynamic system (n=28) or in a static system (n=20) for 4 days. Uncultured 8-day-old (n=9) or 12-day-old (n=17) ovaries served as baseline or in vivo controls, respectively. SETTING: Academic research center. ANIMAL(S): Newborn female BALB/c mice (n=37). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Histologic follicle classification and counting and assessment of follicular viability via immunofluorescent staining. RESULT(S): The percentage of secondary follicles after dynamic culture was identical to the 12-day-old in vivo control. In contrast, after static culture ovaries showed a significantly higher percentage of secondary follicles. For immunofluorescent viability assessment 6.78 follicles per ovary could be isolated after dynamic culture, whereas only 3.8 follicles per ovary could be isolated after static culture. CONCLUSION(S): Dynamic in vitro culture supports physiologic follicular growth initiation, comparable to that observed in vivo. In contrast, accelerated follicular growth was observed after static culture. These findings add additional evidence to the idea that dynamic culture might be a beneficial first step to initiate follicle growth in vitro within the context of fertility preservation.

Effects of viral strain, transgene position, and target cell type on replication kinetics, genomic stability, and transgene expression of replication-competent murine leukemia virus-based vectors.

The limited efficiency of in vivo gene transfer by replication-deficient retroviral vectors remains an obstacle to achieving effective gene therapy for solid tumors. One approach to circumvent this problem is the use of replication-competent retroviral vectors. However, the application of such vectors is at a comparatively early stage and the effects which virus strain, transgene cassette position, and target cell can exert on vector spread kinetics, genomic stability, and transgene expression levels remain to be fully elucidated. Thus, in this study a panel of vectors allowing the investigation of different design features on an otherwise genetically identical background were analyzed with respect to these readout parameters in cultures of both murine and human cells and in preformed tumors in nude mice. The obtained data revealed that (i) Moloney murine leukemia virus (Mo-MLV)-based vectors spread with faster kinetics, drive higher levels of transgene expression, and are more stable than equivalent Akv-MLV-based vectors; (ii) vectors containing the transgene cassette directly downstream of the envelope gene are genomically more stable than those containing it within the 3'-long terminal repeat U3 region; and (iii) the genomic stability of both strains seems to be cell line dependent.

Hypoxia- and radiation-inducible, breast cell-specific targeting of retroviral vectors.

To facilitate a more efficient radiation and chemotherapy of mammary tumours, synthetic enhancer elements responsive to hypoxia and ionizing radiation were coupled to the mammary-specific minimal promoter of the murine whey acidic protein (WAP) encoding gene. The modified WAP promoter was introduced into a retroviral promoter conversion (ProCon) vector. Expression of a transduced reporter gene in response to hypoxia and radiation was analysed in stably infected mammary cancer cell lines and an up to 9-fold increase in gene expression demonstrated in comparison to the respective basic vector. Expression analyses in vitro, moreover, demonstrated a widely preserved mammary cell-specific promoter activity. For in vivo analyses, xenograft tumours consisting of infected human mammary adenocarcinoma cells were established in SCID/beige mice. Immunohistochemical analyses demonstrated a hypoxia-specific, markedly increased WAP promoter-driven expression in these tumours. Thus, this retroviral vector will facilitate a targeted gene therapeutic approach exploiting the unique environmental condition in solid tumours.