Efficient generation of transgenic mice with intact yeast artificial chromosomes by intracytoplasmic sperm injection.
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.
Departamento de Reproducción Animal, INIA, 28040 Madrid, Spain.
Pedro N
PN
Moreira
Patricia
P
Giraldo
Patricia
P
Cozar
Julio
J
Pozueta
Adela
A
Jiménez
Lluís
L
Montoliu
Alfonso
A
Gutiérrez-Adán
D016115:Albinism, Oculocutaneous / Q000235:genetics / Q000517:prevention & control; D000818:Animals; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D018703:Founder Effect; D015870:Gene Expression; D017930:Genes, Reporter; D008543:Melanins / Q000096:biosynthesis; D008677:Metaphase; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D010641:Phenotype; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods
D008543:Melanins; D014442:Monophenol Monooxygenase