Huwe1 is a ubiquitin E3 ligase that plays a role in regulating the DNA damage response, apoptosis, metabolism, and autophagy in various cell types. The objective of this study was to determine the impact of Huwe1 deletion in different tissues by generating transgenic mouse models. We crossed a tissue-specific Cre mouse line with the Huwe1flox/flox mouse line in which exon 11 of the Huwe1 gene is flanked by two loxP sites. When expressed in the same cells, Cre recombinase would excise exon 11, resulting in a frameshift that generates a premature stop codon. By choosing different tissue-specific Cre mouse lines, this method allowed us to create tissue-specific Huwe1 knockout mice. First, lymphocyte-specific Huwe1 knockout mice were generated using a mouse line that expressed Cre recombinase under the Rag-1 promoter. Second, liver-specific Huwe1 knockout mice were created utilizing an Alb-Cre mouse line that expressed Cre recombinase under the albumin promoter. Lastly, inducible whole-body knockout mice were generated through the Cre allele fused to the modified estrogen receptor (ER), which translocates from the cytoplasm to the nucleus upon binding to the estrogen antagonist, tamoxifen. In this Cre-ER model, injection of tamoxifen induced acute deletion of the Huwe1 gene. In all the knockout mouse models, we performed PCR to confirm their genotypes. We also carried out western blot to analyze the depletion of Huwe1 protein in the tissues. We will discuss phenotypes of each tissue-specific Huwe1 knockout mouse model.
Huwe1 is a ubiquitin E3 ligase that plays a role in regulating the DNA damage response, apoptosis, metabolism, and autophagy in various tissues. As an E3 ligase, Huwe1 promotes proteasomal degradation of various protein substrates. However, in vivo roles of Huwe1 remain elusive. The objective of this study was to create Huwe1 knockout mice and to determine the impact of Huwe1 deletion in different tissues. Through the Cre/loxP system, we generated lymphocyte-specific Huwe1 knockout mice, liver-specific Huwe1 knockout mice, and inducible whole-body knockout mice. Genotypes of the resulting mice were confirmed by PCR, and tissue-specific Huwe1 depletion was also validated by Western blots. We will discuss phenotypes of each tissue-specific Huwe1 knockout mouse model.
Doran, G., Balushi, W., Jones, G., & Webb, C. Generation of Tissue-specific Huwe1 Knockout Mice. https://oaks.kent.edu/node/10264
Doran, Gabrielle, Waad Balushi, Grace Jones, and Caitlyn Webb. n.d. “Generation of Tissue-Specific Huwe1 Knockout Mice”. https://oaks.kent.edu/node/10264.
Doran, G., W. Balushi, G. Jones, and C. Webb. Generation of Tissue-Specific Huwe1 Knockout Mice. https://oaks.kent.edu/node/10264.