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Background There are over 17,000 patients in the US waiting to receive liver transplants, and these numbers are increasing dramatically. Significant effort is being made to obtain functional hepatocytes and liver tissue that can for therapeutic use in patients. Blastocyst complementation is a challenging, innovative technology that could fundamentally change the future of organ transplantation. It requires the knockout (KO) of genes essential for cell or organ development in early stage host embryos followed by injection of donor pluripotent stem cells (PSCs) into host blastocysts to generate chimeric offspring in which progeny of the donor cells populate the open niche to develop functional tissues and organs. Methods The HHEX gene is necessary for proper liver development. We engineered loss of HHEX gene expression in early mouse and pig embryos and performed intraspecies blastocyst complementation of HHEX KO embryos with eGFP-labeled PSCs in order to rescue the loss of liver development. Results Loss of HHEX gene expression resulted in embryonic lethality at day 10.5 in mice and produced characteristics of lethality at day 18 in pigs, with absence of liver tissue in both species. Analyses of mouse and pig HHEX KO fetuses confirmed significant loss of liver-specific gene and protein expression. Intraspecies blastocyst complementation restored liver formation and liver-specific proteins in both mouse and pig. Livers in complemented chimeric fetuses in both species were comprised of eGFP-labeled donor-derived cells and survived beyond the previously observed time of HHEX KO embryonic lethality. Conclusions This work demonstrates that loss of liver development in the HHEX KO can be rescued via blastocyst complementation in both mice and pigs. This complementation strategy is the first step towards generating interspecies chimeras for the goal of producing human liver cells, tissues, and potentially complete organs for clinical transplantation.

Additional file 12. Material and methods.

Additional file 8: Table S1. Sequence of the barcoded primer combinations used to amplify the Olfr16 and Hhex genes of the complemented mice.

Additional file 9: Table S2. Sequences of the primers used in the qRT-PCR analysis.

Due to climate change, cattle can experience heat stress more frequently in traditionally temperate, non-tropical environments. Because of this and the fact that most of the world’s inefficient cattle reside in tropical zones, we set out to demonstrate our ability to adapt Angus animals to heat stress through a single gene edit in the prolactin receptor (PRLR). We selected PRLR, because it is known that the SLICK phenotype results from 1 of 3 different mutations in PRLR and therefore is related to heat stress regulation. The overall project was initiated through a partnership between Acceligen and BluePrint Genetics (BPG). BPG harvested 1415 oocytes from a total of 9 ovum pickup rounds from 7 different Angus donors. These oocytes were graded, selected, and put in maturation medium for shipping to Acceligen. Matured oocytes were fertilized in Fert-TALP medium and co-incubated with frozen/thawed semen from 3 different reproductive certified Angus bulls. Consequently, targeted editing was done on a single cell in the zygotes 12h after fertilization by introduction of guide (g)RNA/Cas9 (250ng μL−1 of each) through intracytoplasmic microinjection. Then, treated zygotes (n=1341) were cultured in synthetic oviductal fluid with amino acids (SOFaa) culture medium under a controlled atmosphere until on Day 6, when 277 (20%) grade 1 embryos were selected and 39 returned to Blueprint Genetics for transfer. Because the number of available recipients was limited, only 4 rounds of fresh embryo transfer were completed, and the remaining embryos (n=190) were vitrified for future transfers. Pregnancy checks by ultrasound on Day 30 revealed a 23% (9/39) rate of pregnancy, which decreased to 13% (5/39) by Day 60. In total, 4 animals reached term and delivered healthy calves. Genetic testing of the PRLR target site was done by amplicon sequencing, which showed 3 edited SLICK animals (75%) and one wild-type. Thus, the SLICK zygote editing by microinjection was demonstrated to be an efficient method to produce bovine beef animals and is currently being prepared for regulatory review in multiple countries and commercialization.

We have previously shown that superstimulation of beef donors with a single intramuscular (IM) injection of Folltropin-V (Bioniche Animal Health, Belleville, Ontario, Canada) diluted in a proprietary slow-release formulation (SRF; Bioniche Animal Health) results in in vivo embryo production comparable with the traditional twice-daily IM administration over 4 days (Tribulo et al. 2010 Reprod. Fertil. Dev. 22, 367). Two experiments were designed to evaluate oocyte recovery and in vitro blastocyst production of oocyte donors superstimulated with Folltropin-V, administered in twice-daily injections or diluted in SRF and given by a single injection. In Experiment 1, crossbred beef cows (n = 11) and heifers (n = 5) were randomly allocated to 3 treatment groups in a crossover design (i.e. all animals were treated 3 times, and all treatments were represented in the 3 replicates). All donors had all follicles >8 mm in diameter ablated by ultrasound-guided follicle aspiration on Day 0, received 500 μg of cloprostenol (prostaglandin F2α, Ciclase, Syntex, Argentina), and were treated as follows: Group 1 (multiple FSH): 160 mg of Folltropin-V divided into 4 IM injections administered twice daily (i.e. Days 1 and 2); Group 2 (single FSH): 160 mg of Folltropin-V diluted in 4 mL of 25% SRF and given by a single IM injection in the neck on Day 1; Group 3 (control): no FSH treatment. On Day 4, oocytes were aspirated from all follicles >3 mm in diameter by ultrasound-guided follicular aspiration, classified, matured in TCM-199 supplemented with 0.4% BSA, fertilized in Fert medium, and in vitro cultured in SOF supplemented with 0.4% BSA under oil at 37°C, 5% CO2, 5% O2, 90% N2, and saturated humidity for 7 days. Data were analyzed by ANOVA. The mean (±SEM) numbers of follicles aspirated and oocytes recovered were higher (P