One-step base editing in multiple genes by direct embryo injection for pig trait improvement

The precise and simultaneous acquisition of multiple beneficial alleles in the genome is in great demand for the development of elite pig breeders. Cytidine base editors(CBEs) that convert C:G to T:A have emerged as powerful tools for single-nucleotide replacement. Whether CBEs can effectively mediate C-to-T substitution at multiple sites/loci for trait improvement by direct zygote injection has not been verified in large animals. Here, we determined the editing efficiency of four CBE variants in porcine embryonic fibroblast cells and embryos. The findings showed that hA3A-BE3-Y130F and hA3A-eBE-Y130F consistently resulted in increased base-editing efficiency and low toxic effects in embryonic development. Further, we verified that using a one-step approach, direct zygote microinjection of the CBE system can generate pigs harboring multiple point mutations.Our process resulted in a stop codon in CD163 and myostatin(MSTN) and introduced a beneficial allele in insulin-like growth factor-2(IGF2). The pigs showed disrupted expression of CD163 and MSTN and increased expression of IGF2, which significantly improved growth performance and infectious disease resistance. Our approach allows immediate introduction of multiple mutations in transgene-free animals to comprehensively improve economic traits through direct embryo microinjection,providing a potential new route to produce elite pig breeders.

Generation of ApoE deficient dogs via combination of embryo injection of CRISPR/Cas9 with somatic cell nuclear transfer

Atherosclerotic cardiovascular disease is the leading cause of death in the world which is resulted from complex interactions among multiple genetic and environmental factors （WHO）. Athero- sclerosis is a chronic inflammatory disease characterized by accumulation of lipids in the arterial wall （Gofman and Lindgren, 1950）. Tremendous clinical and experimental efforts have been made to reveal the pathogenesis of the disease. Nevertheless, the mechanism of atherosclerosis is still unclear. A suitable animal model to study metabolic disorders and subsequent atherosclerosis is a necessity. The traditional method by feeding high fat diet to establish animal models of atherosclerosis disease is time- consuming and laborious, and in many circumstances, the pheno- types are not consistent among the individual models.

Research on the appropriate way to transfer exogenous substances into chicken embryos

In biological research, chicken embryos are a classic experimental model for the exploration of the embryonic development and cell differentiation. Transferring exogenous substances into chicken embryos for producing medical antibodies has been widely used in the production practice. However, there are few studies about the effect of the different injection site and dosage on chicken embryos. The aim of this study was to explore the effects of different injection sites and dosages on chicken embryo hatching rate and development, so as to provide a basis for further studies using the chicken embryo model. Freshly laid eggs （Rugao yellow chicken） were injected with different doses of saline at the tip, equatorial plane and the blunt end of the egg shell, respectively. Egg hatching rate was recorded and compared among injection sites and different doses. A trypan blue stain was also injected at the aforementioned sites and the growth of chicken embryos was observed. The SPSS （statistical package for the social science） software was used to analyze the relationship between the chicken eggs hatching rate and the different injection sites or the different dosages. The experimental results showed that there were significant differences on egg hatching rates among the different injection sites and doses （P〈0.05）. The hatchability of the blunt end injection group was significantly higher than that of the other two sites. The egg hatching rate decreased with increased saline doses. The egg hatching rate of the 100 pL saline injection group was higher than the 200 and 300 μL dosage groups. Ultimately, we suggest that the optimal chicken embryo injection process is during early development, at the blunt end site with a dose less than 100 μL to minimize damage to the egg.

In vitro Culture of Early Rabbit Embryos Injected with Foreign Gene and Its Retention

Fertilized rabbit eggs injected with SMTPGH gene were cultured in vitro and retention of theinjected gene was studied using PCR technique and nonradioactive labelling methed. In a mediumof TC199 + 10% FCS, three quarters of the fertilized eggs developed to the blastocyst stage. Noapparent change of the injected gene was found before the 8-cell stage, after which it was eitherintegrated into the chromosome of the host or lost gradually. But finally, the retention rate of theinjected gene should be equal to its integration rate.

Establishing the lower limits of total serum testosterone among Chinese proven fertile men who received treatment of assisted reproductive technology

Testosterone(T)plays a crucial role in spermatogenesis because extremely low levels of intratesticular T lead to correspondingly low serum levels of total T(tT),severe disorders of spermatogenesis,and male sterility.However,there is little consensus on the lower limits of serum tT in proven fertile men undergoing assisted reproductive technology treatments in Chinese or other Asian populations.We aimed to establish the reference range of serum tT based on a population of 868 fertile Chinese men undergoing in vitro fertilization or intracytoplasmic sperm injection and embryo transfer(IVF/ICSI-ET)treatments.We defined a fertile man as having had a live baby with his partner as recorded in our IVF registration system.The lower limits of serum tT were established using a Siemens IMMULITE 2000 chemiluminescent system.The 1st,2.5th,and 5th percentiles and their 95%confidence intervals(CIs)were 3.6(95%CI:2.7–4.1)nmol l−1,4.3(95%CI:4.1–5.0)nmol l−1,and 5.6(95%CI:4.8–5.8)nmol l−1,respectively.Using the linear correlation of serum tT between the Siemens platform and a liquid chromatography–tandem mass spectrometry platform,the calculated lower limits of serum tT were also established for fertile Chinese men undergoing IVF/ICSI-ET treatments,which will benefit the clinical diagnosis and treatment of male infertility during such procedures.