Propagation and phenotypic analysis of mutant rabbits with MSTN homozygous mutation / 生物工程学报

Myostatin gene (MSTN) encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, MSTN-/- homozygous mutants with "double muscle" phenotypic traits and stable inheritance were bred on the basis of MSTN gene editing rabbits, with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits. MSTN-/- primary mutant rabbits were generated by CRISPR/Cas9 gene editing technology. The primary mutant rabbits were mated with wild type rabbits to produce F1 rabbits, whereas the F2 generation homozygous rabbits were bred by half-sibling mating or backcrossing with F1 generation rabbits of the same mutant strain. Sequence analysis of PCR products and its T vector cloning were used to screen homozygous rabbits. The MSTN mutant rabbits with 14-19 week-old were weighed and the difference of gluteus maximus tissue sections and muscle fiber cross-sectional area were calculated and analyzed. Five primary rabbits with MSTN gene mutation were obtained, among which three were used for homozygous breeding. A total of 15 homozygous rabbits (5 types of mutants) were obtained (M2-a: 3; M2-b: 2; M3-a: 2; M7-a: 6; M7-b: 2). The body weight of MSTN-/- homozygous mutant rabbits aged 14-19 weeks were significantly higher than that of MSTN+/+ wild-type rabbits of the same age ((2 718±120) g vs. (1 969±53) g, P < 0.01, a 38.0% increase). The mean cross sections of gluteus maximus muscle fiber in homozygous mutant rabbits were not only significantly higher than that of wild type rabbits ((3 512.2±439.2) μm2 vs. (1 274.8±327.3) μm2, P < 0.01), but also significantly higher than that of MSTN+/- hemizygous rabbits ((3 512.2±439.2) μm2 vs. (2 610.4±604.4) μm2, P < 0.05). In summary, five homozygous mutants rabbits of MSTN-/- gene were successfully bred, which showed a clear lean phenotype. The results showed that the primary breeds were non-chimeric mutant rabbits, and the mutant traits could be inherited from the offspring. MSTN-/- homozygous mutant rabbits of F2 generation could be obtained from F1 hemizygous rabbits by inbreeding or backcrossing. The progenies of the primary biallelic mutant rabbits were separated into two single-allelic mutants, both of which showed a "double-muscle" phenotype. Thus, this study has made progress in breeding high-quality livestock breeds with gene editing technology.

Effect of Sacubitril/Valsartan for alleviating chronic heart failure in elderly patients after acute myocardial infarction / 中华老年医学杂志

Objective To evaluate the effect of Sacubitril/Valsartan for alleviating chronic heart failure(CHF)in elderly patients after acute myocardial infarction(AMI).Methods A total of 87 elderly patients with AMI-induced CHF treated in Heze Shili Hospital from October 2017 to August 2018 were enrolled and randomly divided into the experimental group(n=42)and the control group(n =45).All patients were given standard AMI treatments,and patients in the experimental group were given Sacubitril/Valsartan (100 mg bid)while those in the control group received Valsartan (80 mg qd).After a follow-up of 12 months,levels of N-terminal pro-brain natriuretic peptide(NT-proBNP),left ventricular end-diastolic diameter(LVDd),left ventricular ejection fraction(LVEF),rates of rehospitalization for heart failure and all-cause mortality were compared between the two groups.Results Among the 87 patients,51 patients(58.6％)were male and 36 were female,with an averageage of(67.4 ± 4.0) years.After 12-months of treatment,patients in the experimental group were associated with significantly lower levels of LVDd[(47.86±3.86)mm vs.(50.73±4.39)mm,P＜0.05]and NT-proBNP [(793.43 ± 335.43) ng/L vs.(1 068.44 ± 344.46) ng/L,P ＜ 0.05] and higher levels of LVEF[(53.74 ± 4.08) ％ vs.(44.42 ± 7.41) ％,P ＜ 0.05] than those in the control group.Moreover,the rehospitalization rate for heart failure was markedly higher in the control group than that in the experimental group[15(33.3％)vs.5(11.9％),P＜0.05],while the rate of all-cause mortality was similar between the two groups[2 (4.8％)vs.3(6.7％),P =0.703].Conclusions Compared with Valsartan,Sacubitril/Valsartan can reduce the incidence of CHF after AMI,improve left ventricular function,and reduce the rehospitalization rate due to CHF in elderly patients.

Effect of Sacubitril/Valsartan for alleviating chronic heart failure in elderly patients after acute myocardial infarction / 中华老年医学杂志

Objective@#To evaluate the effect of Sacubitril/Valsartan for alleviating chronic heart failure(CHF)in elderly patients after acute myocardial infarction(AMI).@*Methods@#A total of 87 elderly patients with AMI-induced CHF treated in Heze Shili Hospital from October 2017 to August 2018 were enrolled and randomly divided into the experimental group(n=42)and the control group(n=45). All patients were given standard AMI treatments, and patients in the experimental group were given Sacubitril/Valsartan(100 mg bid)while those in the control group received Valsartan(80 mg qd). After a follow-up of 12 months, levels of N-terminal pro-brain natriuretic peptide(NT-proBNP), left ventricular end-diastolic diameter(LVDd), left ventricular ejection fraction(LVEF), rates of rehospitalization for heart failure and all-cause mortality were compared between the two groups.@*Results@#Among the 87 patients, 51 patients(58.6%)were male and 36 were female, with an averageage of(67.4±4.0)years.After 12-months of treatment, patients in the experimental group were associated with significantly lower levels of LVDd[(47.86±3.86)mm vs.(50.73±4.39)mm, P<0.05]and NT-proBNP[(793.43±335.43)ng/L vs.(1 068.44±344.46)ng/L, P<0.05]and higher levels of LVEF[(53.74±4.08)% vs.(44.42±7.41)%, P<0.05]than those in the control group.Moreover, the rehospitalization rate for heart failure was markedly higher in the control group than that in the experimental group[15(33.3%)vs.5(11.9%), P<0.05], while the rate of all-cause mortality was similar between the two groups[2(4.8%)vs.3(6.7%), P=0.703].@*Conclusions@#Compared with Valsartan, Sacubitril/Valsartan can reduce the incidence of CHF after AMI, improve left ventricular function, and reduce the rehospitalization rate due to CHF in elderly patients.

BLG gene knockout and hLF gene knock-in at BLG locus in goat by TALENs / 生物工程学报

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.

Targeted exogenous EGFP gene editing in caprine fetus fibroblasts by zinc-finger nucleases / 生物工程学报

Gene knockout by ZFNs (zinc-finger nucleases) is efficient and specific, and successfully applied in more than 10 organisms. Currently, it is unclear whether this technology can be used for knocking-out enhanced green fluorescent protein (EGFP) gene in transgenic goats. Here we constructed and used ZFN-coding plasmids to produce genetic knockouts in the cells of cloned fetus produced from donor cells by microinjection of EGFP gene. Following introduced plasmids into caprine primary cultured fetus fibroblasts by electroporation, targeting of a transgene resulted in sequence mutation. Using the flow cytometric analysis, we confirmed the disappearance of EGFP expression in treated cells. Sequence from PCR products corresponding to targeted site showed that insertion of a G into the exon of EGFP resulted in frame shift mutation. These results suggest that ZFN-mediated gene targeting can apply to caprine fetus fibroblasts, which may open a unique avenue toward the creation of gene knockout goats combining with somatic cell nuclear transfer.

Cloning goat producing human lactoferrin with genetically modified donor cells selected by single or dual markers / 生物工程学报

We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.

Production of cloned goats by transfer of nuclei from transfected caprine mammary gland epithelial cells / 生物工程学报

In this study, we evaluated the development potential of caprine mammary gland epithelial cells (CMGECs) after transfection and nuclear transfer into enucleated, ovulated oocytes. We first isolated CMGECs from udders of lactating goats which were transfected with expression plasmid for human lacterrin and selected by G418. Then we chose sixteen neomycin resistant lines and induced them with prolactin for the expression of human lactoferrin checked by Western blotting. The donor cells, expressing human lactoferrin of 75 kD, were fused and activated with enucleated ovulated oocytes. Pronuclear-stage reconstructed embryos were transferred into the oviducts of 16 recipient goats. There were fourteen (87.5%), thirteen (81.3%), and ten (62.5%) pregnancies confirmed pregnant by ultrasound on Day 30, 60, and 90, respectively. Three recipients carried the pregnancies to term and delivered one goat each. Nested PCR-RFLP analysis confirmed that all of the kids were clones of the donor cells. These results demonstrated that CMGECs after transfection remain totipotent for nuclear transfer.