Hongbing Han

One-step generation of myostatin gene knockout sheep via the CRISPR/Cas9 system

11 Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement,College of Animal Science and Technology, China Agricultural University, Beijing 100193, China2 State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China3 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

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

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.

RAPID COMMUNICATION: Generation of knockout sheep via the CRISPR/Cas9 system.

Sheep are an important source of fiber production. Fibroblast growth factor 5 (FGF5) is a dominant inhibitor of length of the anagen phase of the hair cycle. Knockout or silencing of the gene results in a wooly coat in mice, donkeys, dogs, and rabbits. In sheep breeding, wool length is one of the most important wool quality traits. However, traditional breeding cannot accurately and efficiently mediate an advanced genotype into the sheep genome. In this study, we generated 3 knockout sheep via the 1-step clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Sequencing analysis confirmed that mutations in the gene existed in all germ lines of 3 founders: besides the intact sequence, 3 kinds of deletions in the gene (including 5, 13, and 33 bp) were detected. The changes in the primary and senior structure of the FGF5 protein due to the 3 deletions in founders suggested that the FGF5 protein was dysfunctional. In addition, the expression level of intact mRNA in heterozygous individuals decreased compared with the wild types ( < 0.01). Functionally, we discovered that wool length in founders was significantly longer than in wild types ( < 0.05). Collectively, the knockout sheep with the longer wool length phenotype will provide an efficient way for fast genetic improvement of sheep breeding and promote the development of wool industry.

Efficient production of pronuclear embryos in breeding and nonbreeding season for generating transgenic sheep overexpressing TLR4.

BackgroundBrucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons (breeding and non-breeding season) on superovulation and the imported exogenous gene on growth.ResultsIn total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep. Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons (P > 0.05). The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group (P < 0.05). The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.ConclusionsHere we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.

RNAi combining Sleeping Beauty transposon system inhibits ex vivo expression of foot-and-mouth disease virus VP1 in transgenic sheep cells

Foot and mouth disease, which is induced by the foot and mouth disease virus (FMDV), takes its toll on the cloven-hoofed domestic animals. The VP1 gene in FMDV genome encodes the viral capsid, a vital element for FMDV replication. Sleeping Beauty (SB) is an active DNA-transposon system responsible for genetic transformation and insertional mutagenesis in vertebrates. In this study, a conserved VP1-shRNA which specifically targets the ovine FMDV-VP1 gene was constructed and combined with SB transposase and transposon. Then, they were microinjected into pronuclear embryos to breed transgenic sheep. Ninety-two lambs were born and the VP1-shRNA was positively integrated into eight of them. The rate of transgenic sheep production in SB transposon system was significantly higher than that in controls (13.04% vs. 3.57% and 7.14%, P < 0.05). The ear fibroblasts of the transgenic lambs transfected with the PsiCheck2-VP1 vector had a significant inhibitory effect on the VP1 gene of the FMDV. In conclusion, the VP1-shRNA transgenic sheep were successfully generated by the current new method. The ear fibroblasts from these transgenic sheep possess a great resistance to FMDV. The result indicated that RNAi technology combining the “Sleeping Beauty” transposon system is an efficient method to produce transgenic animals.

Tongue Epithelium Cells from shRNA Mediated Transgenic Goat Show High Resistance to Foot and Mouth Disease Virus

Foot and mouth disease induced by foot and mouth disease virus (FMDV) is severe threat to cloven-hoofed domestic animals. The gene 3Dpol in FMDV genome encodes the viral RNA polymerase, a vital element for FMDV replication. In this study, a conserved 3D-7414shRNA targeting FMDV-3Dpol gene was designed and injected into pronuclear embryos to produce the transgenic goats. Sixty-one goats were produced, of which, seven goats positively integrated 3D-7414shRNA. Loss of function assay demonstrated that siRNA effectively knockdown 3Dpol gene in skin epithelium cells of transgenic goats. Subsequently, the tongue epithelium cells from transgenic and non-transgenic goats were infected with FMDV O/YS/CHA/05 strain. A significant decrease of virus titres and virus copy number was observed in cells of transgenic goats compared with that of non-transgenic goats, which indicated that 3D-7414siRNA inhibited FMDV replication by interfering FMDV-3Dpol gene. Furthermore, we found that expression of TLR7, RIG-I and TRAF6 was lower in FMDV infected cells from transgenic goats compared to that from non-transgenic goats, which might result from lower virus copy number in transgenic goats’ cells. In conclusion, we successfully produced transgenic goats highly expressing 3D-7414siRNA targeting 3Dpol gene, and the tongue epithelium cells from the transgenic goats showed effective resistance to FMDV.

Long-term cryopreservation had no adverse effect on viability of embryos and their offspring in sheep.

Cryopreservation has been widely utilized in livestock and human embryos, which allows for storage of worthy embryos for a long period of time, although it is still uncertain as how long embryos can be cryopreserved in liquid nitrogen. The aims of this study were to evaluate the effects of long-term cryopreservation on birth rate of transferred sheep embryos at morula or blastocyst stage, and to investigate growth performance and viability of their offspring. A total of 373 sheep embryos from the same batch, which had been cryopreserved by conventional procedure for 0.5 yr (n=259) or 7.5 yr (n=114), respectively, were transferred to 373 recipient ewes. In parallel, artificial inseminations, acting as controls, were conducted in the same month in both years (n=81 and n=110) that embryo transfers were performed. Results showed that there were no significant differences in birth rate between short-term cryopreservation group (cryopreserved for 0.5 yr in 2003) and long-term cryopreservation group (cryopreserved for 7.5 yr in 2010) either at the morula or blastocyst stage (p>0.05). No specific differences in birth weight were observed among short-term cryopreservation, artificial insemination 1 (performed in 2003), long-term cryopreservation and artificial insemination 2 (performed in 2010) group (p>0.05). The weaning weights were similar between the short-term cryopreservation and long-term cryopreservation group (p>0.05). The mortality rates of the offspring were similar in both groups as well (p>0.05). We concluded that the long-term cryopreservation did not appear to adversely affect birth rate of the embryos, growth performance and viability of their offspring. Our results indicated that the cryopreserved sheep embryos should be stable in liquid nitrogen for at least 7.5 years.

TLR4 overexpression enhances saturated fatty acid–induced inflammatory cytokine gene expression in sheep

Saturated fatty acids (SFAs) can directly stimulate innate immune responses, thereby exacerbating inflammatory aspects of metabolic syndrome. Dietary SFAs act as ligands of Toll-like receptor 4 (TLR4), triggering associated signaling pathways. In this study, we investigated the role of TLR4 in palm oil SFA-associated inflammatory cytokine gene expression in monocytes/macrophages and adipose tissue using TLR4-overexpressing genetically modified sheep. SFA stimulation resulted in upregulation of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), interferon-γ (IFN-γ), and interleukin-10 (IL-10), and TLR4 overexpression enhanced such SFA-induced inflammatory cytokine expression. Moreover, SFAs markedly activated MyD88-dependent signaling, including IL-1 receptor–associated kinase 4 (IRAK4), TNF receptor–associated factor 6 (TRAF6), and nuclear factor-κB (NF-κB). Taken together, our results indicate that TLR4 overexpression enhances the SFA-induced inflammatory response through MyD88-dependent signaling in monocytes/macrophages and adipose tissue.

Different innate immunity and clearance of Salmonella Pullorum in macrophages from White Leghorn and Tibetan Chickens

Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is responsible for the systemic salmonellosis in different breeds of chickens. Macrophages, as host cells, play a key role in the innate immune response following infection with S. Pullorum. In this study, we first generated macrophages from two breeds of chicken (White Leghorn (WL) and Tibetan Chickens (TC)) peripheral blood monocytes in vitro. Then, we showed that the production of interleukin-1β (IL-1β), macrophage inflammatory protein-1β (MIP-1β) and interleukin-10 (IL-10) in lipopolysaccharide (LPS)-treated macrophages was significantly higher compared with the unstimulated cells in TC. LPS triggered only more expression of IL-10 in WL macrophages. Furthermore, macrophages from TC eliminated intracellular bacteria more efficiently than those from WL after S. Pullorum infection at a multiplicity of infection (MOI) 1. In addition, the variation between individuals and sex had the crucial effect on the immune response to LPS and S. Pullorum invasion.