Efficient and accurate homologous recombination in hESCs and hiPSCs using helper-dependent adenoviral vectors.

Low efficiencies of gene targeting via homologous recombination (HR) have limited basic research and applications using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Here, we show highly and equally efficient gene knockout and knock-in at both transcriptionally active (HPRT1, KU80, LIG1, LIG3) and inactive (HB9) loci in these cells using high-capacity helper-dependent adenoviral vectors (HDAdVs). Without the necessity of introducing artificial DNA double-strand breaks, 7-81% of drug-resistant colonies were gene-targeted by accurate HR, which were not accompanied with additional ectopic integrations. Even at the motor neuron-specific HB9 locus, the enhanced green fluorescent protein (EGFP) gene was accurately knocked in in 23-57% of drug-resistant colonies. In these clones, induced differentiation into the HB9-positive motor neuron correlated with EGFP expression. Furthermore, HDAdV infection had no detectable adverse effects on the undifferentiated state and pluripotency of hESCs and hiPSCs. These results suggest that HDAdV is one of the best methods for efficient and accurate gene targeting in hESCs and hiPSCs and might be especially useful for therapeutic applications.

Factors relating to terminally ill cancer patients' willingness to continue living at home during the early phase of home care after discharge from clinical cancer centers in Japan.

OBJECTIVE: To assess the willingness of Japanese terminally ill cancer patients to continue living at home during the early phase of home care after discharge from a Clinical Cancer Center (CCC) in Japan, and to identify factors relating to their willingness to continue living at home. METHODS: A cross-sectional questionnaire survey of a convenient sample of both Japanese terminally ill cancer patients and their caregivers (PFCs) was conducted (n = 294, effective response rate 25.0%). Questionnaires were mailed and medical records were accessed for 73 pairs of respondents, comprising one terminally ill cancer patient and one PFC. RESULTS: At about 10 days after discharge, 64 patients (88%) wished to continue living at home. A hierarchical logistic regression analysis was performed on the data. It was found that the fewer the medical treatments undergone (OR = 0.20, 95% CI: 0.05-0.72), the higher the patients' perception that their condition was consistent with care at home (OR = 2.77, 95% CI: 1.08-8.62) and with their functional well-being (OR = 1.45, 95% CI: 1.08-2.17). In addition, the higher the caregivers' satisfaction with life (OR = 2.37, 95% CI: 1.15-5.77), the more willing patients tended to be to continue living at home. SIGNIFICANT OF RESULTS: The willingness of Japanese terminally ill cancer patients to continue living at home appears to be affected by caregiver status. This indicates a need for discharging facilities to monitor the state of home assistance and to investigate the nature of assistance required for continuing home care.