Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs

Abstract

Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.

Figure 1. Schematic of TALENs targeting the porcine GGTA1 locus and the activity assay.

(A) Schematic of TALEN Set#1. TALEN repeat domains are colored differently to designate the identity of the repeat variable di-residue (RVD). The relationship between RVDs and the cognate-targeted DNA base is NI = A, HD = C, NN = G, NG = T. (B) Schematic of TALEN Set#2. (C) Detection of TALEN activity using luciferase SSA recombination assay. Luciferase activity was increased by 13.1- and 2.2-fold for TALEN Set#1 and Set#2 respectively compared with the control.

Figure 2. TALEN-mediated mutations in the PFFs, and Gal expression in the mutated PFFs.

(A) WT sequence is shown above and TALEN binding sites are underlined. Both deletion and insertion (denoted with “∆” or “+” with the number of base pairs) events are identified. The terms beginning with “×” at the end of each line represent the number of mutations detected by the sequence. (B) Flow cytometric analysis of cells 4, 25,48, 77 and 96 with FITC-conjugated GS-IB4 lectin staining. The horizontal and vertical axes indicate the intensity of fluorescence and the number of events, respectively. The original PFFs of BMI were used as the positive control and 293T cells were as the negative control. The BMI gap was used as the blank control.

Figure 3. Phenotypes of somatic mutation.

(A) Fibroblasts from the cloned pigs were analyzed by FACS with FITC-conjugated GS-IB4 lectin staining. All three pigs were proven to be α 1,3-Gal-negative. (B) Porcine ear fibroblasts stained with FITC-conjugated GS-IB4 were observed under fluorescence microscopy. The original PFFs of BMI were used as the positive control. The scale bar represented 150 μm. (C) The major tissues were cryosectioned and stained with FITC-conjugated GS-IB4. The scale bar represented 250 μm. (D) Immunofluorescence assay. Fibroblasts from cloned pigs were stained with Gal-α1,3Gal-specific mAb M86. The original PFFs of BMI were used as the positive control. The scale bar represents 50 μm. (E) Western blotting for cells of KO piglets and WT pig. β–actin was used as the internal control. (F)Complement-mediated lysis for double KO fibroblasts and WT pig cells. Data shown here are the average of three trials. HIA–HS was used as the negative control. BSA was used as the reagent control.