Analysis of human blastomere chromosomes following nuclear injection into mature mouse oocytes.

Abstract

Objective: The purpose of this study was to develop a new technique for the analysis of complete sets of human metaphase chromosomes from blastomeres using microinjection into mouse oocytes.Design: Prospective pre-clinical study.Materials/Methods: Human blastomeres were obtained from abnormal non-clinical embryos donated for research. Mouse oocytes were obtained from superovulated BALB/c × C57BL/6 (CB6F1) hybrid female mice via standard procedures. Intact blastomere nuclei were aspirated into the lumen of a hydrofluoric acid-treated glass micropipette. Mouse oocytes were steadied with a holding pipette and a small opening was made in the zona pellucida using a non-contact infrared laser system. The nuclear injection pipette was introduced through this opening and into the plasma using a piezo-electric manipulation unit. The blastomere nucleus was gently expelled into the cytoplasm of the mouse oocyte and the tool carefully withdrawn. Oocytes were placed in standard culture for 30 to 60 minutes, activated by brief exposure to 7% ethanol and replaced in culture in media containing colcemid. Oocytes remained in culture for 4 hours to overnight after which time they were individually fixed on glass slides, observed for the presence of condensed chromosomes, stained, and subjected to fluorescent in situ hybridization protocols with human chromosome specific probes.Results: Mouse oocytes survived the procedure at a rate of approximately 80% and injected nuclei could be recovered from lysing oocytes and re-injected. Oocytes could be readily fixed and several high-quality metaphase chromosome sets were obtained that were suitable for cytogenetic analysis. Approximately half of the injected/treated oocytes produced suitable metaphase chromosomes. It was felt that the human material used in this preliminary study was developmentally compromised and that this had a major impact on the success of the procedure.Conclusions: This is the first report of successful human metaphase chromosome production following nuclear injection into mouse oocytes. This technique would seem to provide an alternative to previously published fusion-based techniques and may possibly provide an improvement in efficacy and quality over these techniques. This methodology is currently undergoing further evaluation with developmentally competent discarded material in our laboratory and being directly compared with a fusion-based technique.Supported By: The Institute for Reproductive Medicine and Science of Saint Barnabas. Objective: The purpose of this study was to develop a new technique for the analysis of complete sets of human metaphase chromosomes from blastomeres using microinjection into mouse oocytes. Design: Prospective pre-clinical study. Materials/Methods: Human blastomeres were obtained from abnormal non-clinical embryos donated for research. Mouse oocytes were obtained from superovulated BALB/c × C57BL/6 (CB6F1) hybrid female mice via standard procedures. Intact blastomere nuclei were aspirated into the lumen of a hydrofluoric acid-treated glass micropipette. Mouse oocytes were steadied with a holding pipette and a small opening was made in the zona pellucida using a non-contact infrared laser system. The nuclear injection pipette was introduced through this opening and into the plasma using a piezo-electric manipulation unit. The blastomere nucleus was gently expelled into the cytoplasm of the mouse oocyte and the tool carefully withdrawn. Oocytes were placed in standard culture for 30 to 60 minutes, activated by brief exposure to 7% ethanol and replaced in culture in media containing colcemid. Oocytes remained in culture for 4 hours to overnight after which time they were individually fixed on glass slides, observed for the presence of condensed chromosomes, stained, and subjected to fluorescent in situ hybridization protocols with human chromosome specific probes. Results: Mouse oocytes survived the procedure at a rate of approximately 80% and injected nuclei could be recovered from lysing oocytes and re-injected. Oocytes could be readily fixed and several high-quality metaphase chromosome sets were obtained that were suitable for cytogenetic analysis. Approximately half of the injected/treated oocytes produced suitable metaphase chromosomes. It was felt that the human material used in this preliminary study was developmentally compromised and that this had a major impact on the success of the procedure. Conclusions: This is the first report of successful human metaphase chromosome production following nuclear injection into mouse oocytes. This technique would seem to provide an alternative to previously published fusion-based techniques and may possibly provide an improvement in efficacy and quality over these techniques. This methodology is currently undergoing further evaluation with developmentally competent discarded material in our laboratory and being directly compared with a fusion-based technique. Supported By: The Institute for Reproductive Medicine and Science of Saint Barnabas.