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Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes
Author(s) -
Wang Bin,
Lazaris A.,
Lindenbaum M.,
Stewart S.,
Co D.,
Perez C.,
Drayer J.,
Karatzas C.N.
Publication year - 2001
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.1107
Subject(s) - pronucleus , zygote , biology , blastocyst , microinjection , embryo , microbiology and biotechnology , andrology , reporter gene , green fluorescent protein , genetics , gene , embryogenesis , gene expression , medicine
The introduction of mammalian artificial chromosomes ( AC s) into zygotes represents an alternative, more predictive technology for the production of recombinant proteins in transgenic animals. The aim of these experiments was to examine the effects of artificial chromosome microinjection into bovine pronuclei on embryo development and reporter gene expression. Bovine oocytes aspirated from 2–5 mm size follicles were matured in vitro for 22 hr. Mature oocytes were fertilized in vitro with frozen‐ thawed bull spermatozoa. Artificial chromosome carrying either β‐galactosidase (Lac‐Z) gene or green fluorescence protein (GFP) gene were isolated by flow cytometry. A single chromosome was microinjected into one of the two pronuclei of bovine zygotes. Sham injected zygotes served as controls. Injected zygotes were cultured in G 1.2 medium for 7 days. Hatched blastocysts were cultured on blocked STO cell feeder layer for attachment and outgrowth of ICM and trophectoderm cells. The results showed a high zygote survival rate following LacZ‐ AC s microinjection (74%). However, the blastocyst development rate after 7 days of culture was significantly lower than that of sham injected zygotes (7.5 vs. 22%). Embryonic cells positive for Lac‐Z gene were detected by PCR in three of nine outgrowth colonies. In addition, GFP gene expression was observed in 15 out of 85 (18%) embryos at the arrested 2‐cell stage to blastocyst stage. Six blastocysts successfully outgrew, three outgrowths were GFP positive for up to 3 weeks in culture. We conclude that the methodology for artificial chromosome delivery into bovine zygotes could lead to viable blastocyst development, and reporter gene expression could be sustained during pre‐implantation development. Mol. Reprod. Dev. 60: 433–438, 2001. © 2001 Wiley‐Liss, Inc.