Chen, W., Kubota, S., Seyama, Y.
Notes: To confirm the G to A mutation caused alternative splicing, a minigene containing exon 6 with or without the G to A mutation was amplified. The 2111bp minigene was T/A cloned into the pTargeT™ Mammalian Expression Vector and transfected into COS cells. The RNA was isolated from the transfected cells and analyzed by RT-PCR. The G to A mutation resulted in a truncated RT-PCR product. To confirm the mutation would cause a truncated, inactive protein. A cDNA was constructed that would be the result of the alternative splicing and subcloned into the pTargeT™ Vector. COS cells expressing the mutant had no detectable sterol 27 hydroxylase activity. (1330)