Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos.

Data analysis in somatic cell nuclear transfer (SCNT) research is usually limited to several hundreds or thousands of reconstructed embryos. Here, we report mass results obtained with an established and consistent porcine SCNT system (handmade cloning [HMC]). During the experimental period, 228,230 reconstructed embryos and 82,969 blastocysts were produced. After being transferred into 656 recipients, 1070 piglets were obtained. First, the effects of different types of donor cells, including fetal fibroblasts (FFs), adult fibroblasts (AFs), adult preadipocytes (APs), and adult blood mesenchymal (BM) cells, were investigated on the further in vitro and in vivo development. Compared to adult donor cells (AFs, APs, BM cells, respectively), FF cells resulted in a lower blastocyst/reconstructed embryo rate (30.38% vs. 37.94%, 34.65%, and 34.87%, respectively), but a higher overall efficiency on the number of piglets born alive per total blastocysts transferred (1.50% vs. 0.86%, 1.03%, and 0.91%, respectively) and a lower rate of developmental abnormalities (10.87% vs. 56.57%, 24.39%, and 51.85%, respectively). Second, recloning was performed with cloned adult fibroblasts (CAFs) and cloned fetal fibroblasts (CFFs). When CAFs were used as the nuclear donor, fewer developmental abnormalities and higher overall efficiency were observed compared to AFs (56.57% vs. 28.13% and 0.86% vs. 1.59%, respectively). However, CFFs had an opposite effect on these parameters when compared with CAFs (94.12% vs. 10.87% and 0.31% vs. 1.50%, respectively). Third, effects of genetic modification on the efficiency of SCNT were investigated with transgenic fetal fibroblasts (TFFs) and gene knockout fetal fibroblasts (KOFFs). Genetic modification of FFs increased developmental abnormalities (38.96% and 25.24% vs. 10.87% for KOFFs, TFFs, and FFs, respectively). KOFFs resulted in lower overall efficiency compared to TFFs and FFs (0.68% vs. 1.62% and 1.50%, respectively). In conclusion, this is the first report of large-scale analysis of porcine cell nuclear transfer that provides important data for potential industrialization of HMC technology.

Newborn hearing concurrent genetic screening for hearing impairment-a clinical practice in 58,397 neonates in Tianjin, China.

OBJECTIVE: Newborn hearing screening (NHS) is used worldwide due to its feasibility and cost-efficiency. However, neonates with late-onset and progressive hearing impairment will be missed by NHS. Genetic factors account for an estimated 60% of congenital profound hearing loss. Our previous cohort studies were carried out in an innovative mode, i.e. hearing concurrent genetic screening, in newborns to improve the abilities or early diagnosis and intervention for the hearing defects. In this study, we performed the first clinical practice of this mode in Tianjin city. METHODS: A large cohort of 58,397 neonates, born between December 2011 and December 2012, in 44 hospitals in Tianjin, were screened for 20 hot spot hearing loss associated mutations from GJB2, GJB3, SLC26A4 and MTRNR1(12S rRNA). The data of genetic screening results was comprehensively analyzed with newborn hearing screening (NHS) results. RESULTS: We developed an accurate, high throughput genetic screening method and applied it to a total of 58,397 newborns in Tianjin. 3225 (5.52%) infants were detected to carry at least one mutation allele in GJB2, GJB3, SLC26A4 or MTRNR1. 34 (0.58‰) infants were positive for hearing loss caused by GJB2 or SLC26A4 mutations (homozygote or compound heterozygote). 54(0.93‰) infants are heterozygous of various genes. 109(1.87‰) infants had the pathological mitochondrial DNA mutation. CONCLUSION: Accurate, comprehensive hearing loss associated genetic screening can facilitate genetic counseling and provides valuable prognostic information to affected infants. This united screening mode of this study was a promising clinical practice.

Handmade cloned transgenic piglets expressing the nematode fat-1 gene.

Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been adapted worldwide, but this application is somewhat limited by its relatively low efficiency. In this study, we used handmade cloning (HMC) established previously to produce transgenic pigs that express the functional nematode fat-1 gene. Codon-optimized mfat-1 was inserted into eukaryotic expression vectors, which were transferred into primary swine donor cells. Reverse transcriptase PCR (RT-PCR), gas chromatography, and chromosome analyses were performed to select donor clones capable of converting n-6 into n-3 fatty acids. Blastocysts derived from the clones that lowered the n-6/n-3 ratio to approximately 1:1 were transferred surgically into the uteri of recipients for transgenic piglets. By HMC, 37% (n=558) of reconstructed embryos developed to the blastocyst stage after 7 days of culture in vitro, with an average cell number of 81±36 (n=14). Three recipients became pregnant after 408 day-6 blastocysts were transferred into four naturally cycling females, and a total of 14 live offspring were produced. The nematode mfat-1 effectively lowered the n-6/n-3 ratio in muscle and major organs of the transgenic pig. Our results will help to establish a reliable procedure and an efficient option in the production of transgenic animals.