Production of Transgenic Pigs Mediated by Pseudotyped Lentivirus and Sperm

Sperm-mediated gene transfer can be a very efficient method to produce transgenic pigs, however, the results from different laboratories had not been widely repeated. Genomic integration of transgene by injection of pseudotyped lentivirus to the perivitelline space has been proved to be a reliable route to generate transgenic animals. To test whether transgene in the lentivirus can be delivered by sperm, we studied incubation of pseudotyped lentiviruses and sperm before insemination. After incubation with pig spermatozoa, 62±3 lentiviral particles were detected per 100 sperm cells using quantitative real-time RT-PCR. The association of lentivirus with sperm was further confirmed by electron microscopy. The sperm incubated with lentiviral particles were artificially inseminated into pigs. Of the 59 piglets born from inseminated 5 sows, 6 piglets (10.17%) carried the transgene based on the PCR identification. Foreign gene and EGFP was successfully detected in ear tissue biopsies from two PCR-positive pigs, revealed via in situ hybridization and immunohistochemistry. Offspring of one PCR-positive boar with normal sows showed PCR-positive. Two PCR-positive founders and offsprings of PCR-positive boar were further identified by Southern-blot analysis, out of which the two founders and two offsprings were positive in Southern blotting, strongly indicating integration of foreign gene into genome. The results indicate that incubation of sperm with pseudotyped lentiviruses can incorporated with sperm-mediated gene transfer to produce transgenic pigs with improved efficiency

Rapid calculation method to evaluate thermal management of LED systems based on an improved multi-layer model

An improved laminated structure model of LED systems is proposed. Besides the light-emitting diode (LED) chip, printed circuit board (PCB) substrate, heat sink, etc, additional interface layers are applied to model the non-ideal surface of thermal interface materials (TIMs). And a rapid calculation method is developed for temperature-related calculations. Through comparisons, the method is proved to be valid, and the execution time can be greatly reduced. It is found that the air gap at the TIM surface does hinder heat conduction but the effect is not obvious. The influence of LED chips on system thermal management is comprehensively analyzed. For a single LED chip, the junction temperature shows a linear relationship with its thermal resistance and heat power. The chip size, as well as the aspect ratio between the chip and the PCB substrate, has great effects on the junction temperature. For a multi-chip array, the impact of the chip quantity is suggested to be considered combined with the chip layout. The mutual thermal interaction among LED chips is of most concern, which will become more serious with an increasing number of chips. The work provides a rapid and flexible evaluation method for thermal management of LED systems

COVID-19-associated impact and post-traumatic stress symptoms 39 days after pandemic in a sample of home-quarantined Chinese college students: the mediating effecting of past stressful events, psychological resilience, and social support

Abstract Background During the COVID-19 outbreak, most Chinese college students were home-quarantined to prevent the spread of the virus. COVID-19-associated impact has been shown to be a risk factor for the development of post-traumatic symptoms disorder (PTSD). However, little is known about the psychological processes that mediate this association. This study investigated the association between COVID-19-associated impact and PTSD and examined whether past stressful events, psychological resilience, and social support have mediating effects on this association. Methods The 12,397 valid responses from 31cities in China via an online survey assessed PTSD symptoms, past stressful events, psychological resilience, social support and social-demographic variables. AMOS was used to test the hypotheses of mediating effects. Results On the 39th day of the declared COVID-19 epidemic in China, 6.75% of the surveyed sample showed PTSD symptoms. A positive mediating effect of past stressful events was found between COVID-19-associated impact and PTSD, whereas psychological resilience and social support had negative mediating effects. The fit indices for the path model were found to be significant (β = 0.28, p < 0.001), COVID-19-associated impact indirectly affects the risk of PTSD through mediating pathways (past stressful events → psychological resilience → social support) on PTSD. Conclusions Attention should be paid to the effects of past stressful events of Chinese college students who were home-quarantined during the COVID-19 epidemic, and strategies should also be implemented to improve social support and develop psychological resilience. Trial registration The study was approved by the ethics committee of the Southwest Minzu University

Effects of Hydroxypropyl Methylcellulose (HPMC) on the Reinforcement of Sand by Microbial-Induced Calcium Carbonate Precipitation (MICP)

Microbial-induced calcium carbonate precipitation (MICP) is a new technology used for reinforcing soils through microbial mineralization. However, the existing MICP treatment technology is more suiTable for deeper soils due to its high permeability. In this study, HPMC, a cohesive material combined with Sporosarcina pasteurii-induced calcium carbonate precipitation was used to improve the surface layer of the soil. It was also tested in different contents of bacterial solutions and cementation solutions and with a different number of MICP treatments, and was analyzed and discussed by measuring the surface layer strength, calcium carbonate content generated in the crust, rainfall erosion resistance, wind erosion resistance, and ammonia retention rate of the specimens. The microstructure of the samples was investigated by XRD and SEM. It was shown that the addition of HPMC and increasing the number of MICP treatments were effective in the increase in the calcium carbonate content in the surface crust, but had no effect on the total amount of induced calcium carbonate. The combination of HPMC addition and MICP technology can effectively reduce rainwater scour loss and wind erosion loss, and increase its rainfall erosion resistance and wind erosion resistance. Rainfall losses were reduced by up to 30% in specimens treated with HPMC for MICP technology. When the wind speed was 12 m/s, the maximum mass loss rate of the specimens treated with HPMC for MICP was only 0.828%. The addition of HPMC can effectively improve the ammonia absorption rate and reduce the release of ammonia in the process of MICP technology, which is of great significance for environmental protection. The microstructure showed that the addition of HPMC and the increase in the number of treatments using MICP technology can make the surface structure of the specimens more compact, and the calcium carbonate can more effectively fill the pores and cement the soil particles, while the addition of HPMC may not change the calcium carbonate crystal type

Map of lentiviral vector.

<p>Map of lentiviral vector.</p

Absorption of lentiviral particles by pig spermatozoa under a scanning electronic microscope and immunochemical detection of lentiviral particles on spermatozoa.

<p>A: lentiviral particles (40000×). B: Two lentiviral particles are clearly attached on the head of one spermatozoa (10000×). C: Two lentiviral particles absorbed on a spermatozoa tail (25000×). D: Observation of interaction between a lentiviral particle and the spermatozoa surface in an enlarged image (60000×). E: A lentiviral particle entering a spermatozoon (7500×). Immunuchemical detection of lentiviral particles attached on sperm was performed using a monoclonal mouse anti-VSV-Glycoprotein antibody, F was view field of porcine spermatozoa (200×), and red fluorescence was clearly observed emitted at 570 nm (G, 200×).</p

PCR amplification of transgene DNA sequences in piglets.

<p>PCR amplification of transgene DNA sequences in piglets.</p

Number of lentiviral particles associated with sperm cells after incubation (n = 3).

<p>Number of lentiviral particles associated with sperm cells after incubation (n = 3).</p

RT-PCR amplification of a specific fragment of the lentiviral vector after incubation with sperm cells.

<p>Lane M: DNA Ladder DL2000 (2000, 1000, 750, 500, 250 and 100 bp from top to bottom). Lane 1: Negative control with H₂O as template. Lane 2: Negative control with total RNA extracted from sperm alone as template, both of which showed no PCR products. Lanes 3–6: Specific PCR products were detected in sperm samples incubated with lentiviral particles for 2, 4, 6 and 8 h, respectively.</p