An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving as the mammary gland bioreactor to produce melatonin‐enriched milk in sheep

Abstract

Melatonin as a potent antioxidant exhibits important nutritional and medicinal values. To produce melatonin-enriched milk will benefit the consumers. In this study, a sheep bioreactor which generates melatonin-enriched milk has been successfully developed by the technology that combined CRISPR/Cas9 system and microinjection. The AANAT and ASMT were cloned from pineal gland of Dorper sheep (Ovis aries). The in vitro studies found that AANAT and ASMT were successfully transferred to the mammary epithelial cell lines and significantly increased melatonin production in the culture medium compared to the nontransgenic cell lines. In addition, the Cas9 mRNA, sgRNA, and the linearized vectors pBC1-AANAT and pBC1-ASMT were co-injected into the cytoplasm of pronuclear embryos which were implanted into ewes by oviducts transferring. Thirty-four transgenic sheep were generated with the transgenic positive rate being roughly 35% which were identified by Southern blot and sequencing. Seven carried transgenic AANAT, two carried ASMT, and 25 carried both of AANAT and ASMT genes. RT-PCR and Western blot demonstrated that the lambs expressed these genes in their mammary epithelial cells and these animals produced melatonin-enriched milk. This is the first report to show a functional AANAT and ASMT transgenic animal model which produce significantly high levels of melatonin milk compared to their wild-type counterparts. The advanced technologies used in the study laid a foundation for generating large transgenic livestock, for example, the cows, which can produce high level of melatonin milk.

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

“…Using the HMEJ approach, we observed a similar increase in KI efficiency (37.0%) compared to methods utilizing the traditional HR template (13.8%; ~2.7X increase). These results are similar to efficiencies previously reported in livestock species for KI via cytoplasmic injection of an HR template (5.7% in sheep embryos 16 and 6.5% in pig embryos 17 ). While there have been reports of using genome editors and donor template ODNs to induce single nucleotide polymorphisms or precise deletions of bovine embryos 18,19 , this is the first reported use of the CRISPR/Cas9 system for efficient KI of a large DNA segment (1.8kb).…”

Effects of AANAT overexpression on the inflammatory responses and autophagy activity in the cellular and transgenic animal levels

“…Many melatoninenriched AANAT transgenic plants have been developed and the beneficial effects on these plants have also been reported [22][23][24]80]. In mammals, few AANAT transgenic models have been reported except for one by Ma et al [25]. Compared to the plants, transgenic animals are complex and difficult to be developed.…”

“…These transgenic plants have a high melatonin and low indoleacetic acid content compared to the WT. In contrast, few AANAT transgenic livestock have been reported, except for a study by our group [25]. In the current study, we used state-ofthe-art methodologies to develop AANAT transgenic dairy goats and investigated the impact of AANAT transgenes on the anti-inflammatory activity of the animals.…”

Genome editing approaches to augment livestock breeding programs

“…However, singlestrand oligo DNAs (ssODN) have been used successfully by several groups to make DNA point mutations or small fragment insertions/ deletions by HDR Niu et al, 2018;Wei et al, 2018;Zhou et al, 2016b). Another approach to adding entire genes into zygotes was successfully used to create sheep expressing two proteins involved in the synthesis of the antioxidant melatonin in their milk using a β-casein promoter (Ma et al, 2017). Linear dsDNA without homology arms was injected directly into the cytoplasm of zygotes along with Cas9 and an sgRNA targeting the myostatin locus.…”