Cloning of a gene-edited macaque monkey by somatic cell nuclear transfer

Abstract

Cloning of macaque monkeys by somatic cell nucleus transfer (SCNT) allows the generation of monkeys with uniform genetic backgrounds that are useful for the development of non-human primate models of human diseases. Here, we report the feasibility of this approach by SCNT of fibroblasts from a macaque monkey (Macaca fascicularis), in which a core circadian transcription factor BMAL1 was knocked out by clustered regularly interspaced short palindromic repeat/Cas9 gene editing (see accompanying paper). Out of 325 SCNT embryos transferred into 65 surrogate monkeys, we cloned five macaque monkeys with BMAL1 mutations in both alleles without mosaicism, with nuclear genes identical to that of the fibroblast donor monkey. Further peripheral blood mRNA analysis confirmed the complete absence of the wild-type BMAL1 transcript. This study demonstrates that the SCNT approach could be used to generate cloned monkeys from fibroblasts of a young adult monkeys and paves the way for the development of macaque monkey disease models with uniform genetic backgrounds.

Keywords: CRISPR/Cas9 gene editing; circadian rhythm disorders; macaque monkey cloning; non-human primate models; somatic cell nuclear transfer.

Figure 1.

Preparation of fibroblasts from a BMAL1-edited monkey. (A) A schematic diagram depicting the procedure of culturing fibroblasts. (B) BMAL1-edited founder monkey A6 (see accompanying paper). (C) Primary fibroblast culture derived from the skin of the A6 founder monkey; scale bar, 200 μm. (D) Normal karyotype of A6 fibroblasts. (E) TA clone analysis of the PCR products using DNA (ear tissue, blood cell and fibroblasts) from the A6 monkey. (F) Single-cell genotype analysis of A6 fibroblasts.

Figure 2.

Generation of monkey offspring by SCNT using fibroblasts from a BMAL1 knockout monkey. (A) A schematic diagram depicting the procedure of generating BMAL1 knockout monkey clones. (B) Example images of monkey SCNT embryos at different stages; scale bar, 120 μm. (C) Images of B1–B5, five cloned cynomolgus monkeys generated by SCNT using fibroblasts from the BMAL1 knockout monkey A6. (D) BMAL1 mutation analysis on ear tissue from all five cloned monkeys, showing that four monkeys carry heterogeneous bi-allelic (−8/−8, +4, 2PM) and one carries homogeneous bi-allelic (−8/−8) mutation of the BMAL1 gene.

Figure 3.

Analysis of the genetic origin and BMAL1 expression of cloned monkeys. (A–E) Examples of SNPs for B1–B5, respectively, showing mtDNA SNPs that were identical to those of the oocyte donor monkey, but different from those of surrogate monkey and donor fibroblasts. (F) Examples of STRs from ear tissue samples taken from five cloned monkeys (B1–B5), showing that their nuclear DNA was identical to that of the donor fibroblast, but different from those of their oocyte donor and surrogate monkeys. A more extensive list of the STRs is given in Table S2. (G) Reverse transcription PCR analysis of BMAL1 expression on the blood sample from cloned monkey B1 showed a complete absence of the wild-type BMAL1 transcript.