The chicken hypersensitive site-4 insulator cannot fully shield the murine phosphoglycerate kinase-1 promoter from integration site effects in transgenic mice.

Differential expression of transgenes in transgenic animals is one of the main drawbacks of pronuclear injection. To overwhelm this issue, the genetic constructs are equipped with insulators. In this study, the consensus of exerting chicken hypersensitive site-4 (cHS4) insulator was examined on the shield of phosphoglycerate kinase-1 (Pgk-1) promoter from the surrounding chromatin in transgenic mice. The PGK-EGFP cassette was flanked by insertion of three copies of the cHS4 insulators. Mouse zygotes' microinjection by the constructed cassette was resulted in the birth of nine transgenic founders (F0). Copy-number-dependent expression of the EGFP was investigated in the transgenic F1 offspring by fluorometry and real-time PCR. They showed no correlation between the expression level of transgene and gene copy number among the transgenic lines. Moreover, dissection of the EGFP-expressing mice revealed heterogeneous expression of the EGFP in the different organs. In conclusion, for the first time, these findings indicated that the cHS4 sequence is not a perfect insulator to fully protect the Pgk-1 promoter from the side effects of integration site in transgenic mice and it needs probably to some additional elements of the cHS4 locus to reach this goal.