Fig. 9. Generation of TLR3-null mice. The TLR3 genomic DNA was isolated from the 129/Sv mouse genomic library and characterized by restriction enzyme mapping and sequencing analysis. The targeting vector was constructed by replacing third and fourth exons of the cording region with a neomycin-resistance gene cassette (neo), and a herpes simplex virus thymidine kinase (HSV-tk) was inserted into the genomic fragment for negative selection. The targeting vector was transfected into embryonic stem cells (embryonic day 14.1). G418 and ganciclovir doubly resistant colonies were selected and screened by PCR and Southern blotting. Homologous recombinants were microinjected into C57BL/6 blastocysts. Chimeric mice were mated with C57BL/6 female mice, and heterozygous F₁ progenies were intercrossed in order to obtain TLR3- ^/- mice. Maps of the TLR3 genome, the targeting vector and the predicted disrupted gene are depicted. Restriction enzymes: B, BamHI; E, EcoRI.