206 Towards the generation of transchromosomic goats for the production of fully human immunoglobulin

Abstract

We have previously reported that inactivation of endogenous immunoglobulin (Ig) genes can enhance the production of human polyclonal antibodies (hpAb) in transchromosomic (Tc) cattle containing a human artificial chromosome (HAC) comprising the entire human Ig gene repertoire. Goats offer the advantages of having a shorter gestation period and growing to adult size much faster than larger ungulates. Therefore, based upon our previously established Tc cattle platform, in this study, we aimed to generate immunoglobulin lambda light chain knockout (IGLλ ^−/− )/HAC Tc goats expressing hpAb using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. First, we designed multiple specific single-guide RNAs (sgRNA) targeting the coding sequences of constant regions of goat IGLλ1, IGLλ2 (both in NC_030824.1), and IGLλ3 (NW_017189530.1) and constructed corresponding gene targeting vectors. Gene targeting efficiency analysis in goat fibroblasts showed that the sgRNA were efficient in directing Cas9 to generate targeted indels in corresponding IGLλ genes with efficiencies ranging between 20 and 30%. We then targeted the IGLλ genes and generated a cloned IGLλ ^−/− fetus by SCNT (first round of cloning; G1) using single cell-derived IGLλ ^−/− fibroblast colonies as nuclear donors and established IGLλ ^−/− fetal fibroblast cell lines. Second, we established IGLλ ^−/− /HAC fibroblast colonies by transferring the isKcHACΔ, a HAC containing the entire unrearranged human immunoglobulin heavy-chain and kappa-chain sequences, into the IGLλ ^−/− fetal fibroblast cells via microcell-mediated chromosome transfer (MMCT; Matsushita et al. 2014 PLoS One 9, e90383). The resulting IGLλ ^−/− /HAC fibroblasts were used as nuclear donors for the production of IGLλ ^−/− /HAC goats by SCNT (second round of cloning; G2). A total of 311 one-cell stage G2 embryos were generated using 4 IGLλ ^−/− /HAC fetal fibroblast colonies and surgically transferred into 19 oestrus-synchronized recipients. Seven pregnancies were confirmed (7/19; 36.8%) with ultrasonography at Day 60 ± 3 of gestation, and 1 pregnancy (1/7; 14.3%) developed to term, leading to the birth of a live and healthy kid. Genetic and serological characterisation of this Tc goat produced by the second round of SCNT is in progress.