- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
SLIM microscopy allows for visualization of DNA-containing liposomes designed for sperm-mediated gene transfer in cattle
摘要： Naked DNA has been shown to bind naturally to the sperm, a method called sperm-mediated gene transfer (SMGT). Based on these observations, we examined the efficiency of exogenous DNA binding to sperm using liposomes. In this experiment, we analyzed methods to select frozen-thawed bovine sperm, and evaluated the binding of exogenous DNA to those sperm. To determine the optimal selection method, we used Computer-Assisted Sperm Analysis (CASA). Percoll or Swim-Up were used to select sperm, followed by incubation up to 3 h with the liposome-DNA complexes. The samples were collected after 1 h and after 3 h. We used enhanced green fluorescent protein (eGFP) in combination with the liposomes as a marker for exogenous DNA binding. Five treatments per selection method were analyzed: (1) no incubation, no liposomes and no DNA, (2) incubation with no liposomes and no DNA, (3) incubation with liposomes and no DNA, (4) incubation with liposomes and 1 μg of DNA and (5) incubation with liposomes and 10 μg of DNA. The CASA results for total motility and rapid motility were statistically significant (P < 0.01) between the control and the other treatments in the Percoll group as opposed to Swim-Up. Swim-Up was therefore chosen as the optimal selection method. In order to determine if the liposome-DNA complex had bound to sperm, real time PCR was used to detect GFP DNA and images of the sperm were analyzed using the Spatial Light Interference Microscopy (SLIM). SLIM confirmed the presence of liposomes on the sperm head and tail.
关键词： GFP,Sperm,Liposome,SLIM,Microscopy,Sperm-mediated gene transfer
更新于2025-09-23 15:19:57
-
[IEEE 2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) - Munich, Germany (2019.6.23-2019.6.27)] 2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) - Femtosecond Laser Induced Surface Micro-Structure Building by Material Ejection and Ablation on Cu and Al
摘要： Theoretical and experimental approaches verifying the fluidic operation of a partially shielded digital microfluidics device are presented in this paper. This paper is motivated by recent demand from the synthetic biology community for electrowetting on dielectric (EWD) enabled in-droplet electroporation, but is generalizable to a range of EWD applications that require shielding structures to be patterned on the EWD. An electrode patterned in an additional metal layer on the insulator that supports EWD actuation reduces the effective strength of the EW force due to dielectric shielding at the droplet contact line. A numerical model was developed to predict the impact of the partially shielding electrode on threshold voltage, EW force, fluid velocity, and droplet transport time. Compared with a batch of devices lacking the extra electrode, the presence of the added metal layer resulted in a 29% increase in threshold voltage, an 82% increase in transport time, and a 44% decrease in average transport velocity. Each trend agrees with the simulation results obtained from the fluid transport model. These results support the development of design rules for microfluidic devices that require partially shielding metal layers to integrate with EWD device architectures.
关键词： gene transfer,Fluidics,model,transport,electroporation,electrowetting on dielectric,electrotransfer,digital microfluidics,mechanics,finite element model,device
更新于2025-09-19 17:13:59