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.
Animals ; Base Sequence ; Cloning, Organism ; Electrophoresis ; Endonucleases ; genetics ; metabolism ; Fetus ; Fibroblasts ; metabolism ; Gene Knockout Techniques ; Gene Targeting ; methods ; Goats ; Green Fluorescent Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Zinc Fingers

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.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

Objective To analyze and explore the analgesic effect of Angelica dahurica in neuropathic pain and its regulatory effect on the Mas-related G-protein coupled receptor member D(MrgprD)-transient receptor potential ankyrin 1(TRPA1)signaling pathway,using a mouse model of sciatic nerve chronic constriction injury(CCI).Methods A CCI mouse model was prepared by sterile surgical ligation and wrapping of the sciatic nerve in 30 mice.Pain-related behavioral changes induced by mechanical stimulation were detected by the VonFrey method,and the thermal hyperalgesic effects of Angelica dahurica were evaluated by thermal radiation experiments.The effects of Angelica dahurica on the protein expression levels MrgprD and TRPA1,the number of dorsal root ganglion(DRG)positive neurons,and mRNA levels of MrgprD and TRPA1 in mice were detected by Western Blot,immunofluorescence,and reverse transcription-polymerase chain reaction,respectively.Differences in fluorescence signal intensity in HEK293 cells after single transfection and co-transfection with MrgprD and TRPA1 plasmids,respectively,were analyzed by calcium imaging experiments.Results A total of 25 CCI mouse models were successfully prepared,with a modeling rate of 83.33％(25/30).The mechanical threshold and foot retraction latency were significantly higher in CCI mice treated with Angelica dahurica compared with the control group(P＜0.05).Expression levels of MrgprD and TRPA1 proteins were significantly lower in CCI mice treated with Angelica dahurica than in the control group(P＜0.05).The number of MrgprD-and TRPA1-positive neurons in the DRG was significantly lower group(P＜0.05)and the mRNA levels of MrgprD and TRPA1 were also significantly lower in CCI mice treated with Angelica dahurica than in the control group(P＜0.05).The fluorescence intensity was significantly higher in HEK293 cells co-transfected with MrgprD and TRPA1 plasmids than in single-transfected and blank control cells(P＜0.05).Conclusions This study demonstrated that the MrgprD-TRPA1 pathway is an important target for neuropathic pain,and indicated that Angelica dahurica can inhibit neuropathic pain by regulating this signal transduction pathway.These result provide a foundation for further research on the development of new clinical analgesic drugs and analgesic mechanisms.

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.
Animals ; CRISPR-Cas Systems/genetics* ; Gene Editing ; Muscle, Skeletal/metabolism* ; Mutation ; Myostatin/metabolism* ; Phenotype ; Rabbits

The genetic background such as copy number, integration site and chromosome karyotype of exogenous genes of transgenic animals obtained by random integration is still unclear. There may be some problems such as silent integration, invalid integration, toxic integration and unpredictable expression level of exogenous genes. In this study, six primary (F0) and their corresponding offspring (F1) of human lactoferrin (hLF) transgenic goats were selected as the research objects, and blood samples were collected from jugular vein and DNA were extracted. The genetic background and expression level of exogenous genes were studied by chromosome karyotype analysis, real-time quantitative PCR (qPCR), ELISA and Western blotting. The chromosomes of six F0 transgenic goats had no obvious morphological variation, number change and other abnormalities. The relative copy number was different (2-16) and could be steadily inherited to the next generation. The copy number of F0 and F1 hLF gene was the same. The highest expression level of hLF was 1.12 g/L in F1 transgenic goats (L3-1, 8 copies). The results proved that the integrated exogenous genes could steadily inherit the next generation, and did not cause obstacles to the growth and development of transgenic goat individuals. Moreover, there was no obvious correlation between the number of copies and the expression level of hLF. This laid a foundation for the new varieties cultivation of transgenic goats and other transgenic animals, and analysis of genetic background.