Transgenic analyses. (A) Structure and copy number of the BAC109 transgene in line Tg92. The BAC109 transgene, the relative positions of the Ndn and Magel2 genes, and the transgenic Eag I and Pme I restriction fragments detected with probes 5' and 3' are represented on the upper diagram. Genomic DNA was isolated from wild type (WT) or transgenic (TG) mice, digested by Eag I or Pme I, separated by PFGE, blotted and hybridized to the 5' or 3' probes. The presence of the 54 and 50 kb Eag1 transgenic fragments detected by the 5' and the 3' probes respectively demonstrate the integrity of the transgene. Eag 1 is a methylation sensitive enzyme which explains the presence of a higher hybridization band corresponding to undigested methylated DNA (Und). Hybridization of Pme I digested genomic DNA with either the 5' or 3' Ndn probes, indicated that the BAC integrated in one full copy along with a truncated copy containing the whole 5' region upstream Ndn up to the Ndn promoter.(B) Non imprinted brain-specific expression of transgenic Ndn. Ndn expression was tested by Northern blot analysis. RNAs were isolated from Tg92mat-Ndn+/-pat or wild type littermate tissues: Br, brain, Kd, kidney, Lv, liver, Sp, spleen, Th, thymus, Ht, heart, Mu, muscles. Note that Ndn transgenic mRNAs are not detected in adult muscles whereas Ndn endogenous mRNAs are. After being hybridized to an intragenic Ndn probe, Northern blots were stripped and rehybridized to a β-actin probe to check for the presence and integrity of RNAs.