Cholesterol auxotrophic cells are unable to produce their own cholesterol so they must rely on LDL present in the cell culture media. Please briefly describe three different ways that can be used to create a cholesterol auxotrophic cell line.

The three methods are: 1) Using H-Ras- SREBP- 2 Fusion proteins, the NH2 segment can be released by two sequentia

Explanation:

Sterol regulatory element binding proteins (SREBPs) are transcription factors attached to the endoplasmic reticulum. The NH2-segment, which activates transcription, is connected to membranes by a hairpin anchor formed by two transmembrane sequences and a short lumenal loop. Using H-Ras–SREBP-2 fusion proteins, we show that the NH2-segment is released from membranes by two sequential cleavages. The first, regulated by sterols, occurs in the lumenal loop. The second, not regulated by sterols, occurs within the first transmembrane domain. The liberated NH2-segment enters the nucleus and activates genes controlling cholesterol synthesis and uptake. Certain mutant Chinese hamster ovary cells are auxotrophic for cholesterol because they fail to carry out the second cleavage; the NH2-segment remains membrane-bound and transcription is not activated.

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