The Absence of Calponin 2 in Rabbits Suggests Caution in Choosing Animal Models

Abstract

While the rapid development of CRISPR/CAS9 technology has allowed for readily performing site-specific genomic editing in non-rodent species, an emerging challenge is to select the most suitable species to generate animal models for the study of human biology and diseases. Improving CRISPR/CAS9 methodology for more effective and precise editing in the rabbit genome to replicate human disease is an active area of biomedical research. Although rabbits are more closely related to humans than mice (based on DNA sequence analysis), our whole-genome protein database search revealed that rabbits have more missing human protein sequences than mice. Hence, precisely replicating human diseases in rabbits requires further consideration, especially in studies involving essential functions of the missing proteins. For example, rabbits lack calponin 2, an actin-associated cytoskeletal protein that is important in the pathogenesis of inflammatory arthritis, atherosclerosis, and calcific aortic valve disease. The justification of using rabbits as models for human biomedical research is based on their larger size and their closer phylogenetic distance to humans (based on sequence similarity of conserved genes), but this may be misleading. Our findings, which consider whole-genome protein profiling together with actual protein expressions, serve as a warning to the scientific community to consider overall conservation as well as the conservation of specific proteins when choosing an animal model to study a particular aspect of human biology prior to investing in genetic engineering.

Keywords: CRISPR/Cas9 gene editing; calponin 2; comparative genomics; protein profiling; rabbit.

FIGURE 1

Human calponin isoforms and transgelin. CNN1 (GenBank NC_000019.10, region 11538775.11550323), CNN2 (GenBank NC_000019.10, region 1026608.1039065), and CNN3 (GenBank NC_000001.11, region complement 94896949.94927223) genes each consists of seven exons. The three calponin isoforms are conserved in structure except the C-terminal region that is diverged in length and amino acid sequences as shown in the figure. TAGLN (SM22alpha) (GenBank NC_000011.10, region 117199294.117207465) contains only four coding exons corresponding to the conserved core structure of calponin family. Positions of the CH (calponin homology) domain and two actin binding sites are indicated in the alignment map.

FIGURE 2

Calponin 2 is absent in rabbits. Rabbit tissue lysates were subjected to SDS-PAGE (top panel) and immunoblotting using monoclonal antibodies (mAbs) specific to calponin 1 (CP1) (Jin et al., 1996), calponin 2 (2B8/CP21) (Hossain et al., 2006), calponin 3 (1A4 raised against mouse calponin 3 expressed from cloned cDNA) or SM22alpha/transgelin (3F6) (Liu et al., 2017) (lower panels). While calponin 1 and calponin 2 have similar mobility in the SDS-gel used for Western blotting, calponin 3 and SM22alpha are readily distinguished from calponin 1 and calponin 2 by their molecular weights. Mouse stomach and bladder were used as positive controls. Whereas the expression of calponin 1, calponin 3, and SM22alpha was detected in specific tissue types as previously shown in mice and humans, calponin 2 is completely absent in rabbit tissues as demonstrated in the mAb 2B8/CP21 blot. MHC, myosin heavy chain.

FIGURE 3

Maximum likelihood phylogeny of calponin and transgelin homologs from humans (H), mice (M), and rabbits (R). Mouse proteins are in blue whereas rabbit proteins are in red. The primary entries of the three human calponin isoforms are indicated in parentheses. The branches are supported by bootstrap values of which values greater than 80% are shown.