Sperm genome cloning used in biparental bovine embryo reconstruction
Gabriel Vichera A , Ramiro Olivera A , Pablo Sipowicz B , Martín Radrizzani B and Daniel Salamone A C
+ Author Affiliations
- Author Affiliations
A Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, C1417 Buenos Aires, Argentina.
B Laboratorio de Neuro y Citogenética Molecular, Universidad de San Martín, Av. Gral Paz 5445, B1650 Buenos Aires, Argentina.
C Corresponding author. Email: salamone@agro.uba.ar
Reproduction, Fertility and Development 23(6) 769-779 https://doi.org/10.1071/RD10252
Submitted: 2 October 2010 Accepted: 1 February 2011 Published: 30 June 2011
Abstract
The generation of androgenetic haploid embryos enables several haploid blastomeres to be obtained as identical copies of a single spermatozoon genome. In the present study, we compared the developmental ability of bovine androgenetic haploid embryos constructed by different methods, namely IVF and intracytoplasmic sperm injection (ICSI) before and after oocyte enucleation. Once obtained, the blastomeres of these androgenetic haploid embryos were used as male genome donors to reconstruct biparental embryos by fusion with matured oocytes. To verify the cytoplasmic contribution of androgenetic haploid blastomeres, we used spermatozoa incubated previously with exogenous DNA that coded for a green fluorescent protein gene (pCX-EGFP) and the enhanced green fluorescent protein (EGFP)-positive androgenetic haploid blastomeres generated were fused with mature oocytes. Of the reconstructed embryos reaching the cleavage and blastocyst stages, 85.1% and 9.0%, respectively, expressed EGFP (P > 0.05). EGFP expression was observed in 100% of reconstructed embryos, with 91.2% exhibiting homogenic expression. To confirm sperm genome incorporation, androgenetic haploid blastomeres generated by ICSI prior to enucleation and using Y chromosome sexed spermatozoa were used for biparental embryo reconstruction. Incorporation of the Y chromosome was confirmed by polymerase chain reaction and fluorescence in situ hybridisation analysis. In conclusion, the results of the present study prove that it is possible to use sperm genome replicates to reconstruct biparental bovine embryos and that it is a highly efficient technique to generate homogeneous transgene-expressing embryos.
Additional keywords: sexed haploid NT, sperm cloning, transgenesis.
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