Transgenic mouse strains to investigate biased ghrelin receptor (GHSR1a) drug candidates

Technology

Duke inventors have created five novel transgenic mice with point, knockout, or humanizing mutations to the ghrelin receptor (GHSR1a):

G protein-biased GHSR1a mouse (GHSR-P147A): CRISPR-Cas9 RNA-induced homology-directed repair (HDR) was used to create a missense point mutation within Exon 2 of the endogenous mouse GHSR protein in C57BL/6 ES cells. Specifically, alanine replaced proline at position 147 in intracellular loop-2 to create a protein fully biased to the G protein pathway.

ß-arrestin-biased GHSR1a mouse (GHSR-L148G): CRISPR-Cas9 guide RNA induced HDR was used to create a missesnse point mutation within Exon 2 of the endogenous mouse GHSR protein in C57BL/6 ES cells. Specifically, the mutation caused glycine to replace leucine at position 148 in intracellkular loop-2 to create a fully ß-arrestin-biased GHSR1a protein.

GHSR knockout mouse (GHSR-KO): CIRSPR-Cas9 guide RNA was used to create a directed deletion mutation in Exon 2 of the endogenous mouse GHSR protein. This generated a mouse with a non-functional GHSR protein.

GHSR-ECL2 humanized mouse (GHSR-A199P): CRISPR-Cas9 guide RNA-induced HDR was used to create a missesnse point mutation within Exon 2 of the endogenous mouse GHSR protein in C57BL/6 ES cells. Specifically, the mutation caused a proline residue to replace an alanine at position 199 in extracellular loop-2. This humanized the receptor site.

Humanized GHSR mouse with epitope tag and LoxP insertions (HA-hGHSR-floxed): A humanized GHSR protein sequence replaced the endogenouse mouse GHSR protein in this mouse model. Specifically, the insertion contained three copies of an N-terminal hemagglutinin (HA) tag, the human GHSR gene sequence with introns, and an intronic LoxP site insertion to enable conditional deletion with Cre-driver mice. The human GHSR protein remains under the endogenouse mouse propotor and regulatory elements. These mice allow protein experssion determination by immunolocalization, drug develoment testing, and evaluation of cell/tissue effects of GHSR.