Mouse fibroblast cell line lacking the gene for cytochrome c.
Cytochrome c is a heme protein that is localized in the compartment between the inner and outer mitochondrial membranes where it functions to transfer electrons between complex III and complex IV of the respiratory chain. To generate this cell line, the lab crossed mice harboring a heterozygous somatic cyt c KO allele (cyt cs+/-) with mice harboring a homozygous testis cyt c KO allele (cyt ct-/-). They used homozygous cyt ct-/- females for the crosses. They rescued the embryonic lethal phenotype by introducing a ubiquitously expressed somatic cyt c transgene flanked by loxP sites. Crosses of one of the transgenic animals with the cyt ct-/-cyt cs+/- animals gave rise to F1 mice that when backcrossed produced mice with the cyt ct-/-cyt cs-/- cyt cflox/o genotype, demonstrating that the transgene was able to rescue the embryonic lethal phenotype of the cyt cs KO. Lung fibroblasts (LF) from these animals were cultured and the transgenic cyt c cDNA deleted ex vivo by an adenovirus preparation expressing the Cre recombinase. They isolated individual clones by serial dilution and screened them for the presence of Cyt c. As expected, some of the isolated clones had no detectable Cyt c as determined by immunocytochemistry or western blot. This is one of such KO clones. Retroviral introduction of adenovirus E6/E7 was performed for immortalization. Generation of the line is described in details in PMID: 17210651. All mice were in C57BL6J background.
From the laboratory of Carlos T. Moraes, PhD, University of Miami.
|Product Type:||Cell Line|
|Cell Type:||Lung Fibroblast|
|Biosafety Level:||BSL 1|
|Growth Conditions:||High Glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1 mM pyruvate, 50 ug/ml uridine|
|Subculturing:||Split when confluent|
|Cryopreservation:||Growing media with 50% FBS and 10% DMSO|
|Comments:||E6/E7 immortalized fibroblasts|
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