Effect of DNA loop anchorage regions (LARs) and microinjection timing on expression of beta-galactosidase gene injected into one-cell rabbit embryos.

The conditions favoring expression of a reporter gene microinjected into a male pronucleus of naturally ovulated and fertilized rabbit eggs have been studied. Injection of the reporter gene during S phase of the cell-cycle allows the highest level of expression of the gene. Incorporation of DNA loop anchorage regions (LARs) into constructs upstream and/or downstream of the reporter gene significantly increased the efficiency of expression. In all cases the expression of the microinjected gene started after a period of transcriptional quiescence, i.e., together with the expression of the host genome. Correct targeting of microinjected constructs within the nuclei via interaction of LAR elements with receptor sites on the nucleoskeleton may facilitate expression of injected DNA constructs as well as their integration into host cell DNA.

Heterologous CpG island becomes extensively methylated in the genome of transgenic mice.

It is demonstrated that a heterologous (chicken) CpG island containing five Sp1 canonical recognition sequences becomes highly methylated in the genome of transgenic mice bearing one or several copies of the transgene. Similar levels of methylation of the chicken CpG island were observed in different tissues of transgenic mice except the brain where the level of methylation of this chicken CpG-rich fragment was significantly lower than in other tissues. Analysis of susceptibility of the "transgenic" CpG island to Hpa II and Msp I restriction nucleases revealed an unusual methylation pattern interfering with the action of both of these enzymes. A conclusion has been drawn that heterologous CpG island per se does not contain all necessary signals permitting to maintain its own non-methylated status in the genome of transgenic animals.