378 EFFECT OF TRANSGENE CONCENTRATION AND FLANKING MATRIX ATTACHMENT REGIONS ON THE EFFICIENCY OF ICSI-MEDIATED TRANSGENIC MOUSE PRODUCTION

Abstract

The efficiency of ICSI-mediated transgenesis can be influenced by many different factors. In this study, we have assessed the impact of transgene concentration and of the presence of transgene-flanking matrix attachment regions (MARs) on the efficiency of this procedure. Presented data include assays with three DNA constructs; a plasmid EGFP of 5.4 kb (pEGFP, Clontech Laboratories, Inc., Palo Alto, CA, USA); this plasmid EGFP construct flanked with two MAR elements (M-EGFP-M; 2.3 kb of the human ²-interferon domain boundaries) (Gutiérrez-Adán et al. 2000 Transgenic Res. 9, 81-89); and a yeast artificial chromosome (YAC) of 520 kb. ICSI-mediated transgenesis was done in the B6D2 hybrid mouse strain as previously described (Moreira et al. 2004 Biol. Reprod. 71, 1943-1947), using two concentrations (1 and 15 ng/¼L) of the M-EGFP-M transgene, three concentrations (1, 6, and 15 ng/¼L) of the pEGFP transgene, and three concentrations (2, 3.6, and 5 ng/¼L) of the YAC construct. Embryos were cultured in vitro until the 2-cell stage and transferred into Day 1 pseudopregnant females. Live offspring or Day 14 fetuses were analyzed by PCR for the detection of the genomic presence of the integrated transgene. The proportion of transgenics (PT) obtained with 2, 3.6, and 5 ng/¼L of the YAC construct was 10, 21, and 13% respectively. A Z-test analysis of the data generated with this construct indicated that the number of transgenic offspring, when expressed as a proportion of injected ova (efficiency rate of the procedure, ER), increased with DNA concentration up to a threshold of 3.6 ng/¼L, and suggested that higher concentrations of this transgene (and maybe of co-purifying contaminants) decreased the ER of the procedure. Interestingly, although much higher transgene concentrations were used for the pEGFP construct, this threshold was not reached. With an increase in transgene concentration (from 1 to 15 ng/¼L), the PT increased (from 39 to 70%), as well as the ER of the procedure (from 3 to 7%). In addition, independently of the transgene concentration tested, when M-EGFP-M was used, the ER of the procedure and the PT observed were similar to the ones obtained with pEGFP. These results indicate that, regardless of the importance of other factors such as transgene size, purity, and copy number, the concentration to be used on ICSI-mediated transgenesis should be optimized for each construct, in order to maximize the PT among the liveborn offspring and the overall efficiency of the procedure. Our results also indicate that transgene-flanking MARs do not have a significant impact on the efficiency of this transgenesis method.