No abstract

A recombinant retroviral vector (MFG-GC) was used to study the efficiency of transduction of the human gene encoding glucocerebrosidase (GC; D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45), in mouse hematopoietic stem cells and expression in their progeny. Transfer of the GC gene to CFU-S (spleen cell colony-forming units) in primary and secondary recipients was virtually 100%. In mice 4-7 months after transplantation, highly efficient transfer of the human gene to bone marrow cells capable of long-term reconstitution was confirmed by detection of one or two copies per mouse genome in hematopoietic tissues and in cultures of pure macrophages. Expression of the human gene exceeded endogenous activity by several fold in primary and secondary CFU-S, tissues from long-term reconstituted mice, and explanted macrophages cultures. These studies are evidence of the feasibility of efficient transfer of the GC gene to hematopoietic stem cells and expression in their progeny for many months after reconstitution. The results of this study strengthen the rationale for gene therapy as a treatment for Gaucher disease.

Background: Time-lapse microscopic imaging provides a powerful approach for following changes in cell phenotype over time. Visible responses of whole cells can yield insight into functional changes that underlie physiological processes in health and disease. For example, features of cell motility accompany molecular changes that are central to the immune response, to carcinogenesis and metastasis, to wound healing and tissue regeneration, and to the myriad developmental processes that generate an organism. Previously reported image processing methods for motility analysis required custom viewing devices and manual interactions that may introduce bias, that slow throughput, and that constrain the scope of experiments in terms of the number of treatment variables, time period of observation, replication and statistical options. Here we describe a fully automated system in which images are acquired 24/7 from 384 well plates and are automatically processed to yield high-content motility and morphological data.

Pretreatment of retroviral supernatants with the cationic transgene by polymerase chain reaction (PCR). Colonies liposomes DOTMA-DOPE (Lipofectin), DC-Chol-DOPE from cells infected using centrifugation were positive 27% and DOSPA-DOPE (Lipofectamine) was found to enhance of the time, while the combined approach had positive colstatic transductions of TF-1 target cells. The relative effeconies 31 and 50% of the time for DC-Chol and Lipofectamtiveness at increasing transduction efficiencies (TE) was:ine, respectively. The addition of protamine sulfate to the DOSPA Ͼ DC-Chol Ͼ DOTMA, resulting in average liposome-supernatant mixture during pretreatment was increases over nontreated controls of 11.9-, 6.2-and 1.2-found to be inhibitory. With increasing centrifugal force, the fold, respectively. This pretreatment was found to be syner-TE of cells infected with Lipofectamine pretreated and gistic when combined with centrifugation, having the same untreated supernatants increased proportionally. However, order of effectiveness, and resulting in 57-, 35-and 27-fold the TE of the cells infected with the pretreated superincreases over nontreated controls. For Lipofectamine and natants was significantly higher than the TE of the cells DC-Chol-DOPE liposomes, the combined approach infected with untreated supernatants at all points examyielded 2.2-and 1.3-fold increases over untreated centriined. The increase in TE associated with liposomal prefuged samples. Individual colonies picked from colonytreatment of retroviral supernatants was not shown to be forming unit granulocyte-macrophage assays of infected attributed to a nonreceptor-mediated pathway for viral CD34 + cells were screened for the presence of the entry into the cell.

Smad3 protein is a prominent member of the Tgfb receptor signaling pathway. Smad3(-/-) mice display decreased radiation-induced skin fibrosis, suggesting a defect in both Tgfb-mediated fibroblast proliferation and migration. We established bone marrow stromal cell lines from Smad3(-/-) mice and homozygous littermate(+/+) mice. Smad3(-/-) cells displayed a significant increase in radiation resistance with a D(0)=2.25+/- 0.14 Gy compared to Smad3(+/+) cells with a D(0)=1.75+/- 0.03 (P=0.023). Radioresistance was abrogated by reinsertion of the human SMAD3 transgene, resulting in a D(0)=1.49 0.10 (P=0.028) for Smad3(-/-)(3) cells. More Smad3(-/-) cells than Smad3(+/+) cells were in the G(2)/M phase; Smad3(-/-)(3) cells were similar to Smad3(+/+) cells. Smad3(+/+) cells exhibited increased apoptosis 24 h after 5 Gy (15%) or 8 Gy (43%) compared to less than 1% in Smad3(-/-) cells exposed to either dose. The movement of Smad3(-/-) cells, measured in an automated cell tracking system, was slower than that of Smad3(+/+) cells. Smad3(-/-)(3) cells resembled Smad3(+/+) cells. These studies establish concordance of a defective Tgfb signal transduction pathway, an increased proportion of G(2)/M cells, and radioresistance. The decreased migratory capacity of Smad3(-/-) cells in vitro correlates with decreased radiation fibrosis in vivo in mice deficient in Tgfb signaling.