Down-regulation of cold-inducible RNA-binding protein does not improve hypothermic growth of Chinese hamster ovary cells producing erythropoietin

Discovery of the cold-inducible RNA-binding protein (CIRP) in mouse fibroblasts suggests that growth suppression at hypothermic conditions is due to an active response by the cell rather than due to passive thermal effects. To determine the effect of down-regulated CIRP expression on cell growth and erythropoietin (EPO) production in recombinant Chinese hamster ovary (rCHO) cells at low culture temperature, stable CHO cell clones with reduced CIRP expression level were established by transfecting (rCHO) cells with the CIRP siRNA vector with a target sequence of TCGTCCTTCCATGGCTGTA. For comparison of the degree of specific growth rate (μ) reduction at low culture temperature, three CIRP-reduced clones with different μ and three control clones transfected with null vector were cultivated at two different temperatures, 32 °C and 37 °C. Unlike mouse fibroblasts, alleviation of hypothermic growth arrest of rCHO cells by CIRP down-regulation was insignificant, as shown by statistical analysis using the t-test (P<0.18, n=3). The ratios of μ at 32 °C to μ at 37 °C of CIRP-reduced clones and control clones were 0.29±0.03 and 0.25±0.03 on an average, respectively. Furthermore, it was also found that overexpression of CIRP did not inhibit rCHO cell growth significantly at 37 °C. Taken together, the data obtained show that down-regulation of only CIRP in rCHO cells, unlike mouse fibroblasts, is not sufficient to recover growth arrest at low-temperature culture (32 °C).