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This Article Full Text Full Text (PDF) All Versions of this Article: 71/6/1943    most recent biolreprod.104.032904v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Moreira, P. N. Articles by Gutiérrez-Adán, A. Search for Related Content PubMed PubMed Citation Articles by Moreira, P. N. Articles by Gutiérrez-Adán, A. Agricola Articles by Moreira, P. N. Articles by Gutiérrez-Adán, A.

Efficient Generation of Transgenic Mice with Intact Yeast Artificial Chromosomes by Intracytoplasmic Sperm Injection¹

Departamento de Reproducción Animal,⁵ INIA, 28040 Madrid, Spain Department of Molecular and Cellular Biology,⁶ Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, 28049 Madrid, Spain

The production of animals with large transgenes is an increasingly valuable tool in biotechnology and for genetic studies, including the characterization and manipulation of large genes and polygenic traits. In the present study, we describe an intracytoplasmic sperm injection (ICSI) method for the stable incorporation and phenotypic expression of large yeast artificial chromosomes (YAC) constructs of submegabase and megabase magnitude. By coinjecting spermatozoa and YACs into metaphase II oocytes, we were able to produce founders exhibiting germline transmission of an intact and functional transgene of 250 kilobases, carrying the mouse tyrosinase locus, used here as a reporter gene to rescue the albinism of recipient mice. More than 35% transgenesis was obtained for this YAC transgene. When compared with the pronuclear microinjection standard method, the efficiency of the ICSI-mediated YAC transfer system was significantly greater. In summary, we describe, for the first time, stable incorporation in the host genome and correct phenotypic expression of large DNA constructs mediated by ICSI.

¹ Supported by grants OT02-008 and AGL2002-05783 to A.G.A. and Bio2000-1653 and Bio2003-08196 to L.M. from the Spanish Ministry of Science and Technology.

² Correspondence: Alfonso Gutiérrez-Adán. FAX: +34913474014; agutierr{at}inia.es

³ Correspondence: Lluís Montoliu. FAX: +34915854506; montoliu{at}cnb.uam.es

⁴ Current address: Centro Nacional de Investigaciones Oncológicas, Melchor Fernández Almagro, 3. 28029 Madrid, Spain

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