Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea

Abstract

Neurons of the medial olivary complex inhibit cochlear hair cells through the activation of α9α10-containing nicotinic acetylcholine receptors (nAChRs). Efforts to study the localization of these proteins have been hampered by the absence of reliable antibodies. To overcome this obstacle, CRISPR-Cas9 gene editing was used to generate mice in which a hemagglutinin tag (HA) was attached to the C-terminus of either α9 or α10 proteins. Immunodetection of the HA tag on either subunit in the organ of Corti of adult mice revealed immunopuncta clustered at the synaptic pole of outer hair cells. These puncta were juxtaposed to immunolabeled presynaptic efferent terminals. HA immunopuncta also occurred in inner hair cells of pre-hearing (P7) but not in adult mice. These immunolabeling patterns were similar for both homozygous and heterozygous mice. All HA-tagged genotypes had auditory brainstem responses not significantly different from those of wild type littermates. The activation of efferent neurons in heterozygous mice evoked biphasic postsynaptic currents not significantly different from those of wild type hair cells. However, efferent synaptic responses were significantly smaller and less frequent in the homozygous mice. We show that HA-tagged nAChRs introduced in the mouse by a CRISPR knock-in are regulated and expressed like the native protein, and in the heterozygous condition mediate normal synaptic function. The animals thus generated have clear advantages for localization studies.

Genetic tools for studying cochlear inhibition

“…As Cy3-RgIA5727 will label nAChRs whether native or of viral origin, another tool is needed. Thus, the CRISPR-Cas9 technique was used to produce mice with an HA tag on either the α9 or the α10 subunit of the hair cell nAChR (Vyas et al, 2020). These α9HA, or α10HA mice had no discernible change in hearing (normal ABR thresholds and waveforms), no obvious vestibular deficits (e.g., circling) and growth and breeding appeared normal.…”

1Progress, applications, challenges and prospects of protein purification technology

“…Imagawa et al (2021) purified virus-like particles with FLAGtagged envelope protein through one-step affinity purification with FLAG tag, which could be used as a tetravalent dengue vaccine candidate HA YPYDVPDYA/1.1 kDa The tag is derived from the influenza virus hemagglutinin (Zhou et al, 2012). The tag has little influence on the spatial structure of the target protein, and it is easy to be constructed into tag proteins fused to the N or C terminus Vyas et al (2020) CRISPR-Cas9 gene editing technology was used to connect HA tag as locators to the C-terminus of mouse nicotinic acetylcholine proteins (nAChRs) α9 or α10. The expression of α9 and α10 proteins can be detected…”

Generation of Flag/DYKDDDDK Epitope Tag Knock-In Mice Using i-GONAD Enables Detection of Endogenous CaMKIIα and β Proteins

“…Various commercially available and popular epitope tags have been used for knock-in mouse analysis, including the Flag/DYKDDDDK tag (8 amino acids (AAs)) of synthetic epitope tag [ 13 , 14 , 15 ], HA tag (YPYDVPDYA, 9 AA) of AA residues 98–106 of human influenza hemagglutinin [ 16 , 17 ], Myc tag (EQKLISEEDL, 10 AA) of C-terminal (AA residues 410–419) of human c-myc protein [ 18 ], V5 tag (GKPIPNPLLGLDST, 14 AA or shorter version IPNPLLGLD, 9 AA) of AA residues 95–108 of RNA polymerase alpha subunit of simian virus 5 [ 4 ], and fluorescent tags (GFP, RFP, etc.) [ 4 ].…”

“…Flag tags are commonly used for immunoprecipitation (IP), Western blotting (WB), and immunohistochemistry (IHC) in knock-in mice [ 13 , 14 , 15 ]. In many cases, epitope tag knock-in mice have been created by pronuclear microinjection into fertilized eggs [ 4 , 13 , 14 , 15 , 16 ]. The position of an epitope tag is important for protein function.…”