*4.2. Induction and Assessment of Recombinant Human G1-Induced Arthritis*

To induce arthritis, Nkx2-3−/<sup>−</sup> and BALB/c mice were immunized side-by-side as described previously [5]. Briefly, mice received intraperitoneal injections of 40 μg rhG1 antigen mixed with dimethyl-dioctadecyl-ammonium (DDA) adjuvant dissolved in PBS, on Days 0, 21 and 42. The clinical signs of arthritis were monitored regularly after the second immunization: all mice were examined every second day and the symptoms were quantified using a clinical scoring system ranging from 0 to 4 based on the swelling, redness and ankylosis of the joints of the paws (maximum possible score 16) as described before [4,5]. The diameters of the inflamed front and hind paws and ankles were measured using digital calipers with an accuracy of 0.01 mm two weeks after the third immunization. Three weeks after the last immunization, mice were sacrificed, then blood sera, spleens and lymph nodes were collected for further in vitro studies.
