title: Effect of flanking matrix attachment regions on the expression of microinjected transgenes during preimplantation development of mouse embryos.
abstract: The efficiency of transgenic animal production would increase if microinjected embryos with a successfully integrated transgene could be identified prior to transfer. It is possible to detect microinjected DNA in embryos. However, no reliable system is able to distinguish between transgenes merely present as extrachromosomal DNA and those that have been integrated into chromatin. The experiments reported here were designed to determine if the inclusion of matrix attachment regions (MARs) would enhance the efficiency of transgenic embryos identification using a selection scheme based on the expression of green fluorescent protein (GFP). Pronuclei of mouse embryos were microinjected with GFP reporter gene under the control of three different promoters and flanked or not by three different MAR elements. Transgene expression profiles were followed by direct visualization of GFP in cultured microinjected embryos. Embryos at different developmental stages were classified according to their GFP expression and groups with the same expression pattern were transferred into oviducts of pseudopregnant female mice. Fetuses were collected between days 12-15, and their genomic DNA was purified and analyzed to detect transgene integration. We did not find any statistically significant difference between the percentage of transgenic fetuses produced from GFP-positive or GFP-negative embryos transferred at 4-cell, morula, or blastocyst stage. However, when MAR elements were included in the construct, we found that GFP-positive embryos transferred at the 2-cell stage produced a significantly higher percentage of transgenic fetuses than GFP-negative embryos, but MAR sequences did not completely eliminate false positives.
authors.affiliation: Departamento de Reproducción Animal y Conservación de Recursos Zoogenéticos, INIA, Madrid, Spain. agutierr@inia.es
authors.forename: A
authors.initials: A
authors.lastname: Gutiérrez-Adán
authors.forename: B
authors.initials: B
authors.lastname: Pintado
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D046508:Culture Techniques / Q000379:methods; D003587:Cytomegalovirus / Q000235:genetics; D004624:Embryo Transfer / Q000379:methods; D047108:Embryonic Development; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D008845:Microinjections; D015530:Nuclear Matrix / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D019076:Transgenes / Q000235:genetics*
chemical_list: D017874:Immediate-Early Proteins; D008164:Luminescent Proteins; D010445:Peptide Elongation Factors; D049452:Green Fluorescent Proteins