Figure 1

Generation of transgenic cattle expressing a recombinant anti-CD20 mAb in their milk. (a) Schematic illustration of the two transgene constructs encoding heavy chain (17.2 kb) and light chain (16.5 kb) of the recombinant anti-CD20 mAb, respectively. The transgene construct backbone also contains sequences coding for the following elements: 2× chicken β-globin insulator (2.4 kb); the goat β-casein promoter (4.1 kb); untranslated exon (E)1; parts of E2, E7, E8, and E9 (3.7 kb); and the 3′ genomic DNA of β-casein (5.5 kb). The dotted lines indicate the insertions of the HC- and LC-encoding sequences into the unique XhoI restriction site. The hatched boxes represent the IgG secretory leader sequence, the variable regions (VH and VL), and the constant regions indicated by CH1, Hinge, CH2 and CH3 for HC, or Cκ for LC. The relevant restriction enzymes sites are indicated. (b) PCR detection of the transgenes in transgenic cattle. Marker, 1-kb DNA ladder; P-LC, positive plasmid containing the LC-encoding sequences; P-HC, positive plasmid containing the HC-encoding sequences; WT, genomic DNA of wild-type cattle; genomic DNA of five transgenic cattle lines were indicated by 0802, 1231, 1232, 0216, and 0220. The amplified products for the HC- or LC-encoding sequences were 1.5 kb and 0.8 kb, respectively. (c) Southern blot analysis of transgenic cattle. The digested genomic DNA was hybridized with the probe mixture of the HC- and LC- encoding fragments. The hybridization signals for HC and LC are indicated. P1/P5/P10, signal intensities of the positive plasmid DNA controls equivalent to 1, 5, and 10 gene copies, respectively. (d–f) Expression of the recombinant anti-CD20 mAb in the milk of transgenic cattle, as characterized by SDS-PAGE (d) and western blotting under reducing (e) and non-reducing conditions (f). WT, milk from wild-type cattle. PC, purified human IgG. (g) Recombinant anti-CD20 mAb expression levels in milk from different transgenic cattle were detected by enzyme-linked immunosorbent assays.