Knockdown Clock gene suppressing proliferation of 4T1 cells by inhibiting β-catenin expression

Circadian locomotor output cycles kaput (Clock) gene is a core gene in the circadian rhythm system that is involved in cancer cell proliferation. However, the molecular mechanism of Clock gene participate in the cancer cell proliferation is unclear. Previous studies demonstrated that cell proliferation could be regulated by the canonical Wnt pathway (also known as the Wnt/β-catenin pathway), and the Wnt/β-catenin pathway had a relation with the circadian system. To investigate whether the Clock gene affects the proliferation of breast cancer cell by regulating the expression of β-catenin, we knocked down the Clock expression of mouse breast cancer cells (4T1) by RNA interference. Then detected their proliferation rates using CCK8 assay and the expression of the β-catenin gene by real-time PCR and Western blot. The results showed that the proliferation of the Clock knocked down 4T1 cells is slower than the control. The expression level of β-catenin of these 4T1 cells is reduced. Our study showed that Clock gene knocked down inhibiting the proliferation of the 4T1 cells, probably by suppressing the expression of β-catenin.     

Accumulation of α- and β-globin messenger RNAs in mouse erythroleukemia cells

The accumulation of α- and β-globin mRNA sequences in murine erythroleukemia cells (MELC) treated with various inducers has been studied using specific α- and β-globin complementary DNAs (cDNAs). In cells cultured with dimethylsulfoxide (Me₂SO), hexamethylene bisacetamide (HMBA) or butyric acid, accumulation of α-globin mRNA is detectable after 16, 12 and 8 hr of culture, respectively. An increase in β-globin mRNA sequences is not detected until 20–24 hr after culture. In cells exposed to hemin, both α- and β-globin mRNAs are detectable by 6 hr of culture, and a constant ratio of $\text{α}\text{β}\text{-}\text{mRNA}$ is maintained during induction. In maximally induced cells, the $\text{α}\text{β}\text{-}\text{globin}$ mRNA ratios are approximately 1 in cells induced by Me₂SO and HMBA, and 0.66 and 0.3–0.50 in cells induced by butyric acid and hemin, respectively. Thus different inducers of erythroid differentiation in MELC lead to different times of onset of the expression of α- and β-like genes. In addition, the relative accumulation of α- and β-globin mRNAs in induced cells differs with various types of inducers.     

Linkage of adult α- and β-globin genes in X. laevis and gene duplication by tetraploidization

We have used cloned adult X. laevis α- and β-globin cDNAs to analyze globin genes in X. laevis DNA. We detected α¹- and β¹-globin genes which contain intervening sequences and code for the major adult globins, plus additional diverged α²- and β²-globin genes of unknown coding potential. Unlike the case in mammals, the X. laevis α¹- and β¹-globin genes are closely linked and occur in the sequence 5′-α¹-9 kb-β¹-3′. The α²- and β²-globin genes are also linked, and analysis of globin genes in X. tropicalis suggests that this duplication of an α-β-globin gene pair in X. laevis is the result of chromosome duplication by tetraploidization. The close linkage of α- and β-globin genes in Xenopus provides evidence that vertebrate α- and β-globin genes evolved by tandem duplication of a single primordial globin gene.     

Erythroid-specific expression of β-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34(+) cells with DsRed and a hybrid IHK-β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p=0.05), indicating expression of β-globin from the integrated SB transgene. IHK-β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK-β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK-β-globin transgene for gene therapy of sickle cell disease.     

Transient expression of the β-glucuronidase gene introduced into barley coleoptile cells by microinjection

A -glucuronidase gene was introduced directly into barley (Hordeum vulgare L. cv. Kobinkatagi) coleoptile cells by microinjection and transient expression of the gene was examined. Inner epidermis tissue of coleoptiles was excised and injected with plasmid DNA, pBI221, carrying cauliflower mosaic virus 35S promoter, -glucuronidase gene, and a nopaline synthase polyadenylation region. Histochemical assay for -glucuronidase production showed positive enzyme activity only in coleoptile cells injected with plasmid DNA. Expression of the -glucuronidase gene was examined chronologically using honogenates of injected coleoptile tissues. Glucuronidase activity first appeared after 6 hr, reached the maximum level 24 hr after injection, and decreased afterwards. These results suggest that microinjection of coleoptile tissues may be a useful approach for the genetic engineering of Gramineae plants in which protoplast regeneration is difficult.     

Construction of antisense RNA expression vectors and correction of splicing defect in human β-globin gene (IVS-2-654 C→T mutant) in HeLa cells

The antisense fragments, which were available inin vitro system, were cloned into the mammalian expression vector pcDNA3, and were transfected into H654 cells, a mammalian cell line stably expressing the thalassaemic (IVS-2-654 C→T) human β-globin gene. In these transfected cells, the level of correctly spliced β-globin mRNA in total β-globin mRNA (β/(β + β*)) was improved from 0.07 (0 d) to 0.22 (3 d), and this effect persisted for up to 15 d post transfection. All the results demonstrated that antisense RNAs were able to be transcribed from the antisense fragment expression vectors stably and effectively suppressed aberrant splicing pattern of the mutated β-globin gene (IVS-2-654 C→T) and restored correct splicing pathway. This work provided a novel approach with potential clinical significance to gene therapy of this kind of splicing mutants including β-thalassaemia (IVS-2-654 C→T) by antisense RNAs.     

Absence of messenger RNA and gene DNA for β-globin chains in hereditary persistence of fetal hemoglobin

The relative amounts of α- and β-globin mRNA and globin gene DNA were measured in reticulocyte RNA and lymphocyte DNA of an individual with homozygous hereditary persistence of fetal hemoglobin whose red blood cells contain 100% fetal hemoglobin (Hb F: α₂γ₂). Molecular hybridization assays used as probes full-length DNA copies of human α- and β-globin messenger RNA. The results of these hybridization assays demonstrated the expected amounts of α-globin mRNA and gene DNA, but absence of β-globin mRNA and absence of β-globin gene DNA. In the individual studied, hereditary persistence of fetal hemoglobin is associated with total deletion of the β-globin structural gene.