Effect of a suitable treatment period on the genetic transformation efficiency of the plant leaf disc method.

BACKGROUND: Agrobacterium tumefaciens-mediated leaf disc genetic transformation is an important way to achieve transgenics or gene editing. Ensuring stable and efficient genetic transformation is still an important problem in modern biology. It is assumed that the difference in the development status of genetic transformation cells of receptor materials is the main reason for the difference and instability of genetic transformation efficiency; the stable and efficient genetic transformation rate can be obtained by defining the appropriate treatment period of the receptor material and applying genetic transformation in a timely manner. RESULTS: Based on these assumptions, we studied and established an efficient and stable Agrobacterium-mediated plant transformation system with hybrid poplar (Populus alba × Populus glandulosa, 84 K) leaves, stem segments and tobacco leaves as the research objects. There were differences in the development process of leaf bud primordial cells from different explants, and the genetic transformation efficiency was significantly related to the cell development stage of the in vitro cultured materials. Among them, the genetic transformation rate of poplar and tobacco leaves was the highest on the 3rd and 2nd day of culture, reaching 86.6% and 57.3%, respectively. The genetic transformation rate of poplar stem segments was the highest on the 4th day of culture, reaching 77.8%. The best treatment period was from the development of leaf bud primordial cells to the S phase of the cell cycle. The number of cells detected using flow cytometry and 5-ethynyl-2'-deoxyuridine (EdU) staining, the expression of cell cycle-related protein CDKB1; 2, CDKD1; 1, CYCA3; 4, CYCD1; 1, CYCD3; 2, CYCD6; 1, and CYCH; 1 of explants, and morphological changes of explants can be used as indicators to determine the appropriate treatment period for genetic transformation. CONCLUSIONS: Our study provides a new and universal set of methods and characteristics to identify the S phase of the cell cycle and apply genetic transformation treatments at the appropriate time. Our results are of great significance for improving the efficiency and stability of plant leaf disc genetic transformation.

A Simple Method for Generating, Clearing, and Imaging Pre-vascularized 3D Adipospheres Derived from Human iPS Cells.

Beige/brite/brown-like adipocytes (BAs), dispersed in white adipose tissue, represent promising cell targets to counteract obesity and associated diseases. However, there are major limitations for a BA-based treatment of obesity, among which the main ones are the rareness of BAs in adult humans and the lack of a relevant cell culture condition for modeling the development of BAs. We describe in this chapter the capacity of human induced pluripotent stem cells-derived BA progenitors (hiPSC-BAPs) to self-organize in spheroids and a method for their differentiation at a high efficiency in hiPSC-derived 3D adipospheres containing UCP1-expressing cells. Enrichment of adipospheres with human dermal microvascular endothelial cells (HDMECs) allows to better mimic native adipose tissue. To observe the accumulation of lipid droplets, organization of the extracellular matrix and expression of adipogenic markers on the surface of hiPSC-adipospheres, we detail how to combine Oil Red O staining with immunostaining both imaged by fluorescence microscopy. Furthermore, to have a global view of pre-vascularized network formed by HDMECs inside of hiPSC-adipospheres, we describe a method which consists of the whole adipospheres fixation, multicolor immunostaining, clearing, and imaging.

Whole-Mount Immunostaining for the Identification of Histone Modifications in the S-Phase Nuclei of Arabidopsis Roots.

This chapter describes a method used to analyze the behavior of histone modifications in S phase in Arabidopsis using a whole-mount immunostaining technique. Previous studies have demonstrated that dramatic changes in local chromatin structure are required for the initiation and progression of DNA replication, and that histone modifications play an essential role in the determination of chromatin structure in S phase. Since euchromatic and heterochromatic regions are replicated in distinct S-phase stages, it is important to identify histone modifications at each stage. Here, we introduce a protocol for whole-mount immunostaining combined with 5-ethynyl-2'-deoxyuridine (EdU) staining, which enables the visualization of spatial patterns in histone modifications in the early and late S-phase nuclei of Arabidopsis roots.

MicroRNA-377-3p targeting MMP-16 inhibits ovarian cancer cell growth, invasion, and interstitial transition.

BACKGROUND: To evaluate role of microRNA (miRNA)-377-3p on the remission of ovarian cancer (OC) cell proliferation, invasion, and interstitial transition in vivo and vitro. METHODS: SKOV3 cells were used as the object of in vitro research and four-week-old immunodeficient BABL/c female nude mice were used to form the xenograft model. Cell models were constructed by transfecting NC mimics, miR-377 mimic, plasmid cloning DNA (pcDNA), pc-matrix metalloproteinase (MMP)-16, or co-transfecting miR-377 mimic and pc-MMP-16. TargetScan software was used to predict the targeting relationship between miRNA-377-3p and MMP-16 in OC cells. The combination of miRNA-377-3p and MMP-16 was detected by dual luciferase report experiment. miRNA expression levels of miRNA-377-3p and MMP-16 in each transfection group cells were detected by reverse transcription-polymerase chain reaction (RT-PCR). The proliferation of SKOV3 cells were assessed by 5-ethynyl-2'-deoxyuridine (EdU) staining and microtubule formation, while the invasion ability of SKOV3 cells was detected by Transwell assay. Protein expression levels of MMP-16, survivin, Ki67, vascular endothelial growth factor (VEGF), E-cadherin, and N-cadherin were detected by Western blot (WB), and the positive cells of Ki67 and VEGF were detected by immunohistochemistry (IHC). RESULTS: MMP-16 overexpression markedly increased the EDU-positive cell percentage, upregulated survivin and Ki67 levels, increased the number of invasive cells per field, and enhanced VEGF and N-cadherin expression. Importantly, co-transfection of miRNA-377-3p and MMP-16 reversed these abnormal phenomena. Xenotransplantation mouse models were formed by injecting SKOV-3 cells subcutaneously. Tumor size, tumor volume, and tumor weight were all reduced in the miR-377-3p mimic-transfected group. The results of IHC indicated that Ki67 and VEGF expression were decreased in the miR-377-3p mimic-transfected group. CONCLUSIONS: These findings indicate that miR-377-3p could be a promising therapeutic agent for OC cell growth, invasion, and interstitial transition with MMP-16 being its likely target.

A quantitative analysis of stem cell homeostasis in the Arabidopsis columella root cap.

The Lugol's staining method has been widely used to detect changes in the maintenance of stem cell fate in the columella root cap of Arabidopsis roots since the late 1990s. However, various limitations of this method demand for additional or complementary new approaches. For instance, it is unable to reveal the division rate of columella root cap stem cells. Here we report that, by labeling dividing stem cells with 5-ethynyl-2'-deoxyuridine (EdU), the number and distribution of their labeled progeny can be studied so that the division rate of stem cells can be measured quantitatively and in addition, that the progression of stem cell progeny differentiation can be assessed in combination with Lugol's staining. EdU staining takes few hours and visualization of the stain characteristics of columella root cap can be performed easily under confocal microscopes. This simple technology, when used together with Lugol's staining, provides a novel quantitative method to study the dynamics of stem cell behavior that govern homeostasis in the Arabidopsis columella root cap.