Effect of flanking matrix attachment regions on the expression of microinjected transgenes during preimplantation development of mouse embryos

Key words: GFP, MAR, preimplantation embryo, selection, transgenicAbstractThe efficiency of transgenic animal production would increase if microinjected embryos with a successfully in-tegrated 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 DNAandthose thathave beenintegratedintochromatin. Theexperimentsreportedherewere designedto determineif theinclusion of matrix attachment regions (MARs) would enhance the efficiency of transgenic embryos identificationusing a selection scheme based on the expression of green fluorescent protein (GFP). Pronuclei of mouse embryoswere microinjected with GFP reporter gene under the control of three different promoters and flanked or not bythree different MAR elements. Transgene expression profiles were followed by direct visualization of GFP incultured microinjected embryos. Embryos at different developmental stages were classified according to their GFPexpression and groups with the same expression pattern were transferred into oviducts of pseudopregnant femalemice. Fetuses were collected between days 12–15, and their genomic DNA was purified and analyzed to detecttransgene integration. We did not find any statistically significant difference between the percentage of transgenicfetuses 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 atthe 2-cell stage produced a significantly higher percentage of transgenic fetuses than GFP-negative embryos, butMAR sequences did not completely eliminate false positives.IntroductionPronuclear microinjection of foreign DNA is the mostcommonly used technique to produce transgenic ani-mals (Gordon et al., 1980). Unfortunately, with thisprocedure only about 10–20% of mice born from mi-croinjected embryos are transgenic. The efficiency infarm animals is lower, varying from 0.1 to 4.45%(Pursel & Rexroad, 1993). Due to the high costs andthe long gestation periods of large animals, attemptshave been made to detect transgene integration prior