# Immunodeficient Rabbit Models: History, Current Status and Future Perspectives
## 4 The Birth of Immunodeficient Rabbits
They initially worked to produce IgH-transgenic rabbits carrying a productive VDJ-C\(\upmu\) sequence. Instead of obtaining B-cells producing the immunoglobulins coded by the recombinant VDJ-C\(\upmu\), they found that these transgenic rabbits were B-cell deficient, with a 50-100% reduction in serum IgM and IgG levels. Nevertheless, the production of this ID rabbit line was accidental, unexpected, untargeted, and not reproducible. The first report of ID rabbit production through targeted gene editing, and also the first report of GTT rabbit production in a broader scope, came in 2011, two years after ZFN appeared in the scientific toolbox. [PERSON] and colleagues from the Technical University of Munich, Roache Diagnostics GmBH, and Sangamo BioSciences reported the production of immunoglobulin M (IgM) knockout rabbits by microinjecting ZFN mRNAs to pronuclear stage rabbit embryos [61]. They demonstrated that ZFN efficiently disrupted the target gene, in ex vivo cultured embryos, and ultimately in offspring kits born after embryo transfer. The IgM knockout rates were high, ranging from 23 to 35%, calculated as the number of positive KO founders per born kits. The KO rates were magnitudes higher than those of prior efforts of gene targeting in rabbit cells, which were in the range of \(<\) 0.1% to \(<\) -1% (unpublished data and personal communications). They further demonstrated that the ZFN-introduced mutant alleles transmitted to the next generation animals, satisfied the golden standard of a successful GTT method. Lastly, they showed that gene knockout of IgM, as predicted, led to serum IgM and IgG deficiency and lack of IgM+ and IgG+ B lymphocytes in homozygous KO animals. The success of [PERSON] et al. opened a new possibility in biomedical research, i.e., efficient production of GTT rabbits. The entire research community is no longer bound by the lack of rbESCs to achieve this goal. Nevertheless, it did not take long before the second-generation gene-editing tool TALEN came into play. Compared to ZFN, TALEN has several significant advantages: design and construction are easier, and the efficiency is comparable and often higher. In 2013 Dr. [PERSON]'s group in Guangzhou China obtained _Rag1/2_ KO rabbits by embryo microinjection of TALENs mRNA, the first (and the only to date) report using TALEN for ID rabbit production [60]. The KO efficiency in founders was exceptionally high, reaching 94% for _Rag1_ and 100% for _Rag2_, representing a 3 to 4-fold higher efficiency than those in the ZFN IgM work. The authors found that a high percentage of founder animals carried bi-allelic modifications (61% for _Rag1_; 100% for _Rag2_). As a result, the founder-generation of RAG deficient rabbits displayed typical ID phenotypes with defective T and B cell populations. Only one year after the first TALEN report, [PERSON]'s group used the third-generation tool CRISPR/Cas9 and first produced _Il2 rg_ knockout rabbits [88]. Again, the KO efficiency reached 100% such that all kits born after microinjection/embryo transfer carried mutant alleles. The authors then demonstrated the multiplex capacity of CRISPR/Cas9 by producing _Il2 rg/Rag1_ double knockout rabbits in one round of microinjection/embryo transfer. Comparing to TALEN, the CRISPR/Cas9 system is easier to set up, faster, cheaper, and has similar or higher targeting efficiencies. These favorable traits soon made it the dominant tool in ID rabbit production. In 2017, [PERSON]'s group at the University of Michigan produced multiple lines of ID rabbits, including FOXN1, RAG2, IL2 RG, and PRKDC knockouts by multiplex sgRNA microinjection and multiplex embryo transfer [58]. In the multiplex sgRNA microninjection approach, different sgRNAs targeting different genes were pooled and microinjected to the same embryo, leading to the production of founder rabbits carrying multiple gene knockouts. One such animal named \"NuSRG\" had mutations in all these genes: Foxn1 (_nude_), Prkdc (_SCID_), Rag2 and II2 Rg. This NuSRG rabbit was hairless and severely immunodeficient.