The Neomycin Resistance Cassette in the Targeted Allele of Shank3B Knock-Out Mice Has Potential Off-Target Effects to Produce an Unusual Shank3 Isoform

Chunmei Jin; Hyojin Kang; Taesun Yoo; Jae Ryun Ryu; Ye-Eun Yoo; Ruiying Ma; Yinhua Zhang; Hyae Rim Kang; Yoonhee Kim; Hyunyoung Seong; Geul Bang; Sangwoo Park; Seok-Kyu Kwon; Woong Sun; Hyunkyung Kim; Jin Young Kim; Eunjoon Kim; Kihoon Han

doi:10.3389/fnmol.2020.614435

The Neomycin Resistance Cassette in the Targeted Allele of Shank3B Knock-Out Mice Has Potential Off-Target Effects to Produce an Unusual Shank3 Isoform

Front Mol Neurosci. 2021 Jan 11:13:614435. doi: 10.3389/fnmol.2020.614435. eCollection 2020.

Authors

Chunmei Jin^{1

2}, Hyojin Kang³, Taesun Yoo⁴, Jae Ryun Ryu⁵, Ye-Eun Yoo⁶, Ruiying Ma^{1

2}, Yinhua Zhang^{1

2}, Hyae Rim Kang^{1

2}, Yoonhee Kim¹, Hyunyoung Seong¹, Geul Bang^{7

8}, Sangwoo Park⁷, Seok-Kyu Kwon⁹, Woong Sun^{2

5}, Hyunkyung Kim^{2

10}, Jin Young Kim⁷, Eunjoon Kim^{4

6}, Kihoon Han^{1

2}

Affiliations

¹ Department of Neuroscience, College of Medicine, Korea University, Seoul, South Korea.
² Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, South Korea.
³ Division of National Supercomputing, Korea Institute of Science and Technology Information, Daejeon, South Korea.
⁴ Center for Synaptic Brain Dysfunctions, Institute for Basic Science, Daejeon, South Korea.
⁵ Department of Anatomy, College of Medicine, Korea University, Seoul, South Korea.
⁶ Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea.
⁷ Research Center for Bioconvergence Analysis, Korea Basic Science Institute, Ochang, South Korea.
⁸ College of Pharmacy, Korea University, Sejong, South Korea.
⁹ Center for Functional Connectomics, Korea Institute of Science and Technology, Brain Science Institute, Seoul, South Korea.
¹⁰ Department of Biochemistry and Molecular Biology, College of Medicine, Korea University, Seoul, South Korea.

Abstract

Variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3), which encodes postsynaptic scaffolds, are associated with brain disorders. The targeted alleles in a few Shank3 knock-out (KO) lines contain a neomycin resistance (Neo) cassette, which may perturb the normal expression of neighboring genes; however, this has not been investigated in detail. We previously reported an unexpected increase in the mRNA expression of Shank3 exons 1-12 in the brains of Shank3B KO mice generated by replacing Shank3 exons 13-16 with the Neo cassette. In this study, we confirmed that the increased Shank3 mRNA in Shank3B KO brains produced an unusual ∼60 kDa Shank3 isoform (Shank3-N), which did not properly localize to the synaptic compartment. Functionally, Shank3-N overexpression altered the dendritic spine morphology in cultured neurons. Importantly, Shank3-N expression in Shank3B KO mice was not a compensatory response to a reduction of full-length Shank3 because expression was still detected in the brain after normalizing the level of full-length Shank3. Moreover, in another Shank3 KO line (Shank3 gKO) with a similar Shank3 exonal deletion as that in Shank3B KO mice but without a Neo cassette, the mRNA expression levels of Shank3 exons 1-12 were lower than those of wild-type mice and Shank3-N was not detected in the brain. In addition, the expression levels of genes neighboring Shank3 on chromosome 15 were altered in the striatum of Shank3B KO but not Shank3 gKO mice. These results suggest that the Neo cassette has potential off-target effects in Shank3B KO mice.

Keywords: Neo cassette; Shank3B knock-out; gene expression; mouse chromosome 15; off-target effect.