Regulatory role of energy metabolism in skeletal development

To test the consequence of reduced glycolysis and reduced cytoplasimic acetyl-CoA in mouse skeletal devleopment. To reduce glycolysis, Ldha was homozygously deleted in collagen 2 expressing chondrocytes using the Cre-lox sytem [Col2-Cre:Ldha(fl/fl)] on top of one allele germline deletion of Ldhb [Ldhb(+/-)]. To reduce cytoplasmic/nuclear acetyl-CoA, Acly that catalizes conversion of citreate into acetyl-CoA was deleted in chondrocytes using Col2-Cre. Both mutant mice develop skeletal dysplasia, but former model survive postnatally, whereas Acly mutants die at birth. Overall design: To test the consequence of reduced glycolysis and reduced cytoplasimic acetyl-CoA in mouse skeletal devleopment, we deleted Ldha and one allele of Ldhb, and Acly in chondrocytes. Primary chondrocytes were isolated from P5 Col2-Cre:Ldha(fl/fl):Ldhb+/- mice, and from E18.5 Col2-Cre:Acly(fl/fl) mice. Rib cartilage was digested with 0.2% collagenase in DMEM with 10% FCS. Collagenase was removed and cells were plated at a confluent density. After overnight culture in vitro, RNA was extracted for RNA-seq analysis.