Generation Of B Cell-deficient Pigs By Efficient TALENs Or CRISPR/Cas9-mediated Gene Targeting

Background Polyclonal antibodies that can be used in clinical medicine are in desperately short supply because they are mainly obtained from human donors. However, collecting antibodies from humans is restricted by limitations in the volume of donated blood that can be obtained, the risk of transmission of infectious agents, and ethical concerns relating to primary immunization with certain antigens to human donors. Thus, animal models for the development humanized polyclonal antibodies(hp Abs) are urgently needed. Because of their anatomic, physiological and immunological similarities to humans, pigs could be used as animal models for generating humanized polyclonal antibodies with little ethical concerns.Previous researchers have reported that inactivation of the endogenous immunoglobulin gene(s) can disrupt productive VDJ rearrangement at the HC locus, which further causing depletion of B cells and endogenous Igs in the blood. Besides, as reported, the inactivation of the endogenous Igs can enhance the production of hp Abs by exogenous human genes or cells in animal models. Thus, to generate pig models for hp Abs production, the first step is to generate Ig HC mutated and B celldeficient pigs.Screening targeted cell with gene mutations by homologous recombination combined with SCNT was usually used to construct targeted pig models however, it was time-consuming because of the low targeting efficiency of traditional homologous recombination. In recent years, transcription activator-like(TAL) effector nucleases(TALENs) and The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated(Cas) system(CRISPR/Cas9 system) have emerged as powerful and efficient tools for gene editing and have already been used widely. In this study, we decided to take advantage of TALENs and CRISPR/Cas9 system to disrupt the function of the porcine Ig M gene and produce B cell-deficient pigs and lay a foundation for the generation of pigs that can expresses fully hp Abs.Objective To generate Ig M heavy chain mutated and B cell-deficient pigs models with immunodeficiency disorders.Methods 1. We selected the Ig M JH5 region as the target site, TALENs and CRISPR/Cas9 system plasmid(Cas9-sg RNA) were designed and constructed based on the target site sequence according to their design principles. 2. TALENs or Cas9 plasmid were transfected into PFFs with a certain portion of neomycin-expression plasmid(p CMV-td Tomato) by using nucleofection program. After G418 selection, single colonies were screened and collected for PCR sequencing. Cell colonies with biallelic mutation were obtained. 3. With the aid of somatic cell nuclear transfer(SCNT) technology, cell pools with biallelic mutation were used as nuclear donors to produce cloned Ig M gene mutant piglets. 4. For cloned piglets, PCR sequencing was used to identify their genotypes; Western Blot was taken for the detection of Ig M protein expression; Flow cytometry was performed to analyze Ig M-positive B cells among their PBMCs; the organizational structure of the major secondary lymphoid organs, i.e., mesenteric lymph nodes was detected by HE staining; Immunohistochemistry was alsoperformed on mesenteric LNs to detect T and B cell markers; sandwich ELISA was used to detect the levels of Igs in the plasma of pigs.Results 1. According to the Ig M gene sequence, two pairs of TALEN plasmids were successfully designed and constructed. SSA-EGFP-reporter plasmid was used to detect the targeting ability of TALEN plasmids. Flow cytometry analysis showed the in vitro targeting efficiency of the two TALEN pairs were 40.6% and 46.8%, respectively. In PFFs, the ratios of cell colonies with Ig M mutations in all screened colonies were 7.2% and 15.1% after transfecting the two TALEN pairs respectively. Two cell colonies with biallelic mutation were collected. 2. Cas9-sg RNA plasmid was also designed and constructed to target the Ig M JH5 region. The targeting ability of the Ig M Cas9-sg RNA vector was first tested with ‘Surveyor’ treatment, which indicated that targeting efficiency of the constructed Ig M Cas9-sg RNA plasmid was about 29.3%. We then transfected the plasmid into early passage male primary PFFs and screened cell colonies with Ig M gene mutations. The targeting efficiency of the CRISPR/Cas9 system on PFFs in our study reached 53.3%, and the biallelic mutation rate was 13.3%. Six cell colonies with biallelic mutation were obtained. 3. By applying the SCNT technology, two groups of cloned pigs were constructed and six or five recipient gilts were transferred with Ig M gene mutated reconstructed embryos respectively. In the first six gilts, two were found to be pregnant. Of the two pregnant recipients, one was terminated at 35-days to collect fetuses for PFF isolation and genotype analysis. Nine fetuses were collected. The other recipient was maintained until term and delivered three live male piglets and two mummified corpses. Also two of the other five recipient gilts were pregnant. Twelve 35-day fetuses were collected from one of the recipient gilt(2-4#) and the2-5# recipient delivered five healthy male piglets. 4. The genotypes of the cloned piglets were all detected as Ig M gene mutant and the mutation patterns were in accord with the donor cells. To detect the Ig M protein expression of these gene-mutation piglets, Western blot was performed using full protein extraction of spleen tissue. Compared to the WT piglet, the three cloned piglets had no Ig M heavy chain and less light chain detected. Flow cytometry analysis showed that Wild-type piglets possessed more than 40% Ig M-positive B cells among their PBMCs, in contrast, Ig M-positive B cells were barely present in Ig M-deficient neonatal piglets. Furthermore, histological examination of LNs showed showed a complete lack of follicular structure and germinal center organization in the mesenteric LNs and a lack of CD79a+ B cells and follicular architecture in the cloned piglets. Concomitantly, sandwich ELISA exhibited extremely low levels of Ig M and Ig A in the plasma of the cloned litters when compared with WT piglets and the significantly smaller amounts of Ig G, which would come from breast-feeding milk, could be detected in the plasma of Ig M-knockout piglets compared to WT piglets.Conclusion Our study indicates that the TALENs or CRISPR/Cas9 system combined with SCNT technology is an efficient approach for one-step generating Ig M gene homozygous mutated and B cell-deficient pigs with immunodeficiency disorders. These pigs are also a first significant step towards generating a pig that expresses fully human, antigen-specific polyclonal Ab to target numerous incurable infectious diseases with high unmet clinical need.