MSTN modification in goat mediated by TALENs and performance analysis.

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. It can inhibit the proliferation of myoblasts and serve as an important candidate gene for animal breed improvement. Mutations of the MSTN gene can cause extensive skeletal muscle hyperplasia and hypertrophy, resulting in "double muscle" symptoms. This leads to reduction of animal fat differentiation and increase of muscle content, thereby meeting the demand for quality consumption of animal meat in the market. In order to obtain a double-muscle phenotype using mutant MSTN gene in cloned goat, the goat MSTN gene was target-modified by TALENs. In this study, the TALENs expression vector was designed and constructed in the first exon sequence of the goat MSTN gene, which was then transfected into the goat fetal fibroblasts. The resistant cell lines were obtained by puromycin selection, and the cell lines with the MSTN gene mutations were analyzed by PCR and gene sequencing, thereby identifying the mutation type(s). The MSTN gene mutant cell lines were used as the nuclear donor cells in somatic cell nuclear transfer procedures in goats, and The morphological structure of the muscle tissue of the goats with MSTN gene mutations was analyzed by tissue section. The body weight of the cloned goats were monitored at different months of age, which provided the growth trend of their weight at different developmental stages. The results show that a total of 109 MSTN gene mutant cell lines were obtained. The mutation efficiency was 79.0% (109/138), of which 46 were biallelic mutations, accounting for 33.3% (46/138) of the total cell lines. Four MSTN gene mutant cell lines (1 biallelic homozygous mutation, 3 non-homozygous mutations) with good growth status were selected for somatic cell nuclear transfer in 12 recipients, of which 4 were pregnant by B-ultrasound at 30 days, indicating the a 33.3% (4/12) pregnancy rate. Two cloned goats were born at the end of the pregnancy. Sequencing analysis showed that there was no mutation in one allele of the M-1 cloned lamb, and the other allele harbored a 3 bp-deletion. The M-2 cloned lamb harbored a 1 bp base insertion in one allele of the MSTN gene, and a deletion of 13 bp in the other allele, resulting in mutations in both alleles and the loss of the protein-coding sequence of MSTN after the mutation site. In addition, the muscle fibers of cloned M-1 goats are tightly arranged and thick, and their monthly body weight is higher than that of normal wild-type goats. However, it is still consistent with the growth trend of normal wild-type goats and the M-1 goats can develop into healthy adults. In summary, this study showed that goat fetal fibroblasts with the multiple MSTN gene mutations were successfully obtained by TALENs technology, and cloned goats with mutant MSTN genes could be generated by somatic cell nuclear transfer method, thereby providing a technical foundation for the cultivation of the "double muscle" phenotype goats, and serving as a reference method for the preparation of other transgenic animals in the future.

Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary-specific expression of human lactoferrin in transgenic mice.

It is very important to develop an effective, specific, and robust expression cassette that ensures a high level of expression in the mammary glands. In this study, we designed and constructed a series of mammary gland-specific vectors containing a complex hybrid promoter/enhancer by utilizing promoter sequences from milk proteins (i.e., goat ß-casein, bovine αs1-casein, or goat ß-lactoglobulin) and cytomegalovirus enhancer sequences; vectors containing a single milk protein promoter served as controls. Chicken ß-globin insulator sequences were also included in some of these vectors. The expression of constructs was analyzed through the generation of transgenic mice. Enzyme-linked immunosorbent assay (ELISA) analysis revealed that the hybrid promoter/enhancer could drive the expression of recombinant human lactoferrin (rhLF) cDNA at high levels (1.17-8.10 mg/ml) in the milk of transgenic mice, whereas control promoters achieved a very low rhLF expression (7-40 ng/ml). Moreover, the expression of rhLF was not detected in the serum or saliva of any transgenic animal. This result shows that all constructs, driven by the hybrid promoter/enhancer, had high mammary gland-specific expression pattern. Together, our results suggest that the use of a hybrid promoter/enhancer is a valuable alternative approach for increasing mammary-specific expression of recombinant hLF in a transgenic mouse model.

[Investigation on soil - transmitted nematode infections in national surveillance sites in Jiangsu Province from 2006 to 2015].

OBJECTIVE: To understand the status of soil-transmitted nematode infections in rural residents so as to provide the evidence for formulating the guidance for prevention and control of the diseases. METHODS: The national surveillance sites of soil-transmitted nematode infections were established in Shuyang County, Suqian City, Jiangsu Province from 2006 to 2015. At least 1 000 fecal samples of residents aged 3 years or above were collected in every autumn, and the intestinal helminth eggs were detected with the Kato-Katz technique and the Enterubius vermicularis eggs were detected by the cellophane tape method for children aged 3-12 years. The soil samples were collected from vegetable fields, lavatories, courtyards and kitchens to examine Ascaris lumbricoides eggs and larvae of hookworm. RESULTS: The infection rates of soil-transmitted nematodes in residents and E. vermicularis in children reduced from 1.81% (19/1 049) and 4.72% (5/106) in 2006 to 0.25% (3/1 180) and 0 (0/263) in 2015, respectively, in the surveillance sites. The infection intensity was mild in all the infected cases. The soil samples were negative for detecting A. lumbricoides eggs and hookworm larvae. CONCLUSIONS: The infection rates of soil-transmitted nematodes in the residents and E. vermicularis in the children show a decreasing trend and keep at a low level of prevalence in Shuyang County.

Generation of human lactoferrin transgenic cloned goats using donor cells with dual markers and a modified selection procedure.

The objective was to use dual markers to accurately select genetically modified donor cells and ensure that the resulting somatic cell nuclear transfer kids born were transgenic. Fetal fibroblast cells were transfected with dual marking gene vector (pCNLF-ng) that contained the red-shifted variant of the jellyfish green fluorescent protein (LGFP) and neomycin resistance (Neo) markers. Cell clones that were G418-resistant and polymerase chain reaction-positive were subcultured for several passages; individual cells of the clones were examined with fluorescence microscopy to confirm transgenic integration. Clones in which every cell had bright green fluorescence were used as donor cells for nuclear transfer. In total, 86.7% (26/30) cell clones were confirmed to have transgenic integration of the markers by polymerase chain reaction, 76.7% (23/30) exhibited fluorescence, but only 40% (12/30) of these fluorescent cell clones had fluorescence in all cell populations. Moreover, through several cell passages, only 20% (6/30) of the cell clones exhibited stable LGFP expression. Seven transgenic cloned offspring were produced from these cells by nuclear transfer. Overall, the reconstructed embryo fusion rate was 76.6%, pregnancy rates at 35 and 60 days were 39.1% and 21.7%, respectively, and the offspring birth rate was 1.4%. There were no significant differences between nuclear transfer with dual versus a single (Neo) marker (overall, 73.8% embryo fusion rate, 53.8% and 26.9% pregnancy rates, and 1.9% birth rate with five offspring). In conclusion, the use of LGFP/Neo dual markers and an optimized selection procedure reliably screened genetically modified donor cells, excluded pseudotransgenic cells, and led to production of human lactoferrin transgenic goats. Furthermore, the LGFP/Neo markers had no adverse effects on the efficiency of somatic cell nuclear transfer.