Type-A spermatogonia first appear at between 3-7 d postnatally in mice and are the only immortalized diploid cells that reproduce in adulthood in these animals. In our current study, we explored the feasibility of producing stable transgenic mice using these cells. Enhanced pEGFP-N1 plasmids were suspended in ExGen500 transfection reagent and injected at different angles into the testes of 7-d-old male ICR mice. The resulting type-A spermatogonia-mediated gene transfer （TASMGT） mice were then mated with normal females at different stages of sexual maturity （6, 12, and 24 wk）. The integration and expression of the introduced EGFP gene was evaluated in the F1 transgenic offspring by PCR and Southern blotting analysis. The foreign gene integration rates for a low-dose group （15 μL gene suspension injected into each testis） and a high-dose group （30 μL suspensions injected） at the three stages of female sexual maturity tested were 11.76% （2/17）, 14.29% （3/21）, and 11.11% （2/18）, and 5% （1/20）, 5.56% （1/18）, and 0 （0/17）, respectively. The average integration rates for these two dose groups were 12.5% （7/56） and 3.64% （2/55）, respectively, which was a significant difference （P0.05）. Semi-quantitative RT-PCR analysis further showed that the introduced GFP gene was expressed in 3/9 integration mice. In addition, GFP expression was observed in the sperm cells from the TASMGT mice, and also in the embryos and F2 pups from the F1 generation transgenic mice. Hence, although the foreign gene integration rate for TASMGT is not high and the transgenic offspring show as yet unexplained defects, our results indicate that this method is a potentially feasible and reproducible new approach to creating transgenic mice.
JU Hui-mingBAI Li-jingREN Hong-yanMU Yu-lianYANG Shu-linLI Kui
[Objective] This study aimed to clone the porcine growth hormone gene promoter and determine the core promoter sequences and the cis-acting elements. [Method] Sequence of the 5＇flanking region of porcine growth hormone gene was searched out and downloaded from the NCBI website. According to the targeted se- quence, primers were designed and synthesized for the PCR amplification. The 1 882 bp （-1 821 bp-＋61 bp） fragment was amplified by PCR. Nine promoter frag- ments with different lengths were obtained by genome-walking deletion method and then cloned into luciferase reporter vectors. Relative transcriptional activities of these 5＇ terminal-deleted plasmids in pituitary and non-pituitary cells were determined by transient transfection of the rat pituitary adenoma cell （GH3）, porcine lilac endotheli- um cell （PIEC） and porcrne Kidney-15 （PK15） with the constructed dual-luciferase vectors. [Result] Result of DNA sequencing showed that the 1 882 bp fragment of GH 5＇ promoter was successfully cloned. Nine luciferase reporter gene plasmids were constructed. DuaI-Luciferase reporter assay indicated that the promoter inserted into reporter gene vector had very strong cell specificity. [Conclusion] Porcine growth hormone gene specifically expresses in pituitary cells. The minimal promoter of the porcine growth hormone gene is mapped at the region -110 bp-＋61 bp. Promoter regions 218 bp--110 bp and -429 bp--218 bp contain positive regulatory elements.
Beef and mutton production has been aided by breeding to integrate allelic diversity for myostatin(MSTN),but a lack of diversity in the MSTN germplasm has limited similar advances in pig farming.Moreover,insurmountable challenges with congenital lameness and a dearth of data about the impacts of feed conversion,reproduction,and meat quality in MSTN-edited pigs have also currently blocked progress.Here,in a largest-to-date evaluation of multiple MSTN-edited pig populations,we demonstrated a practical alternative edit-site-based solution that overcomes the major production obstacle of hindlimb weakness.We also provide long-term and multidomain datasets for multiple breeds that illustrate how MSTN-editing can sustainably increase the yields of breed-specific lean meat and the levels of desirable lipids without deleteriously affecting feed-conversion rates or litter size.Apart from establishing a new benchmark for the data scale and quality of genome-edited animal production,our study specifically illustrates how gene-editing site selection profoundly impacts the phenotypic outcomes in diverse genetic back-grounds.
Messenger RNA-like non-coding RNAs （mlncRNAs） are a newly identified group of non-coding RNAs （ncRNAs） that may be involved in a number of critical cellular events. In this study, 93 candidate porcine mlncRNAs were obtained by computational prediction and screening, among which 72 were mapped to the porcine genome. Further analysis of 8 representative candidates revealed that these mlncRNA candidates are not highly conserved among species. Remarkably, one of the candidates, sTF35495, was found to be precursor of a putative porcine microRNA. By RACE PCR, we determined that the full length of sTF35495 was 3 kb. The protein-coding potential of this RNA was tested in silico with no significant finding. Semi-quantitative RT-PCR analysis of the subgroup of 8 candidates revealed two distinct expression profiles and two molecules were further validated by real-time PCR. The predicted pre-microRNA sequence in this study provides a potentially interesting insight into the in vivo function of porcine mlncRNAs and our findings suggest that they play key biological roles in Sus scrofa.
Bang XiaoXingju ZhangYong LiZhonglin TangShulin YangYulian MuWentao CuiHong AoKui Li
