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Matrix attachment regions (MARs) are DNA fragments with specific motifs that enhance transgenic expression; however, the characteristics and functions of these elements remain unclear. In this study, we designed and synthesized three short chimeric MARs, namely, SM4, SM5, and SM6, with different numbers and orders of motifs on the basis of the features and motifs of previously reported MARs, namely, SM1, SM2, and SM3, respectively. Expression vectors with six synthetic MARs flanking the down or upstream of the expression cassette for enhanced green fluorescence protein (EGFP) were constructed and introduced into Chinese hamster ovary (CHO) cells. Results indicated that the EGFP expression of the CHO cells with transfection bySM4, SM5, or SM6-containing vectors was higher than that of those containing SM1, SM2, or SM3 regardless of the MAR insertion position. The improving effect of SM5 was particularly pronounced. Transgenic expression was further enhanced with the increasing SM5 copy number. Bioinformatics analysis indicated that several arrangements of the DNA-binding motifs for CEBP, FAST, Hox, glutathione, and NMP4 may help increase transgenic expression levels and the average population of highly expressed cells. Our findings on novel synthetic MARs will help establish stable expression systems in mammalian cells.

CHO cells are the preferred host for the production of complex pharmaceutical proteins in the biopharmaceutical industry, and genome engineering of CHO cells would benefit product yield and stability. Here, we demonstrated the efficacy of a Dnmt3a‐deficient CHO cell line created by CRISPR/Cas9 genome editing technology through gene disruptions in Dnmt3a, which encode the proteins involved in DNA methyltransferases. The transgenes, which were driven by the 2 commonly used CMV and EF1α promoters, were evaluated for their expression level and stability. The methylation levels of CpG sites in the promoter regions and the global DNA were compared in the transfected cells. The Dnmt3a‐deficent CHO cell line based on Dnmt3a KO displayed an enhanced long‐term stability of transgene expression under the control of the CMV promoter in transfected cells in over 60 passages. Under the CMV promoter, the Dnmt3a‐deficent cell line with a high transgene expression displayed a low methylation rate in the promoter region and global DNA. Under the EF1α promoter, the Dnmt3a‐deficient and normal cell lines with low transgene expression exhibited high DNA methylation rates. These findings provide insight into cell line modification and design for improved recombinant protein production in CHO and other mammalian cells.

In this paper, we report the extension of an earlier developed Direct Numerical Simulation (DNS) model to study coupled heat and mass transfer problems in particulate flows. The DNS model builds on an efficient ghost-cell based Immersed Boundary Method (IBM) which implicitly incorporates the boundary conditions into the discretized momentum, thermal and species conservation equations of the fluid phase. On the particles an exothermic surface reaction takes place. The heat and mass transport is coupled through the particle temperature, which offers a dynamic boundary condition for the fluid phase thermal energy equation. The present simulations are performed for four fluid-solid systems. Following the case of the unsteady mass and heat diffusion in a large pool of quiescent fluid, we consider a stationary sphere under forced convection. In both cases variable reaction rates are imposed at the particle surface, and the particle temperatures obtained from DNS show a good agreement with analytical/empirical solutions. After that, we apply our DNS model to the three-bead reactor and finally, a dense particle array consisting of hundreds of particles distributed in a random fashion is studied. The concentration and temperature profiles are compared with a ID heterogeneous reactor model and the heterogeneity inside the array is discussed. .

In this paper, an efficient ghost-cell based immersed boundary method is introduced to perform direct numerical simulation (DNS) of particulate flows. The fluid-solid coupling is achieved by implicit incorporation of the boundary conditions into the discretized momentum, thermal and species conservation equations of the fluid phase. Taking the advantage of a second order quadratic interpolation scheme, different boundary conditions could be realized consistently in our ghost-cell based immersed boundary method. The heat and mass transport in a fluid-particle system is coupled through the solid temperature, which offers a dynamic boundary condition for the fluid thermal equation. The present simulations are performed for three different fluidsolid systems. The first one is the unsteady mass and heat diffusion in a large pool of quiescent fluid. The solution of the solid temperature development obtained from DNS is compared with the “exact” solution obtained from a standard second-order finite difference technique. Following that, we consider a stationary sphere under forced convection. The steady state temperature of the particle can be calculated from the fluidsolid mass and heat transfer rates, which are obtained from the well-known empirical Ranz-Marshall and Frössling correlations. The last simulation case is an in-line array of three spheres, the so-called three-bead reactor. The computed adiabatic temperature rise obtained from DNS shows good agreement with the value calculated from the overall species conversion ratio of the reactor.

During cardiac development, the growth, remodeling and morphogenesis of embryonic hearts are closely linked to hemodynamic forces. An understanding of the interaction mechanism between hemodynamic forces and heart development is important for the early diagnosis and treatment of various congenital defects. The myocardial wall strain (MWS) in embryonic heart is a critical parameter for quantifying the mechanical properties of cardiac tissues. Here, we focus on the radial strain which is defined as the change of the myocardial wall thickness. An effective measurement of MWS is conductive to studies of embryonic heart development. Chick embryo is a popular animal model used for studing the cardiac development due to the similarity of cardiac development between the human heart and the chick heart at early developmental stages and its easy access. Although various imaging methods have been proposed, there still remain significant challenges to imaging of early stage chick embryo heart because it is small in size and beats fast. Optical coherence tomography (OCT) is a non-contact three-dimensional imaging modality with high spatial and temporal resolution which has been widely used for imaging the biological tissue. In this paper, we describe a method to measure in vivo MWS of chicken embryonic hearts with a high speed spectral domain OCT(SDOCT) system worked at 1310 nm. We perform four-dimensional (4D) (x, y, z, t) scanning on the outflow tract (OFT) of chick embryonic hearts in a non-gated way. The transient states of the OFT are extracted from the 4D data by using the beating synchronization algorithm. The OFT center line can be achieved by image processing. Assuming that the blood flow is parallel to the center line in the blood vessel, we calculate the Doppler angle of blood flow from the OFT center line. In a certain OFT cross-section, the OFT myocardial wall (inner and external borders) is segmented from the OCT images with a semi-automatic boundary-detection algorithm. Then, the myocardial wall thickness is calculated from the Doppler angle, area and sum of inner and external radii of the segmented myocardial wall. The radial strain is obtained by calculating the myocardial wall thickness variation. Previous methods calculated the myocardial wall thickness by directly subtracting inner and external radii. The measured result may be deteriorated by insufficient resolution of the system since the myocardial wall of OFT is very thin. The present method can solve this problem by calculating the thickness through using the sum of the radii instead of the subtraction. The experimental results on embryonic chick hearts demonstrate that the proposed method can measure the MWS of OFT along arbitrary orientation and it is a useful tool for studying the biomechanical characteristics of embryonic hearts.