Generation of Mouse Model of Hemophilia A by Introducing Novel Mutations, Using CRISPR/Nickase Gene Targeting System.

Developing mouse models of hemophilia A has been shown to facilitate in vivo studies to explore the probable mechanism(s) underlying the disease and to examine the efficiency of the relevant potential therapeutics. This study aimed to knockout (KO) the coagulation factor viii (fviii) gene in NMRI mice, using CRISPR/Cas9 (D10A/nickase) system, to generate a mouse model of hemophilia A. Two single guide RNAs (sgRNAs), designed from two distinct regions on NMRI mouse FVIII (mFVIII) exon 3, were designed and inserted in the pX335 vector, expressing both sgRNAs and nickase. The recombinant construct was delivered into mouse zygotes and implanted into the pseudopregnant female mice's uterus. Mutant mice were identified by genotyping, genomic sequencing, and mFVIII activity assessment. Two separate lines of hemophilia A were obtained through interbreeding the offspring of the female mice receiving potential CRISPR-Cas9-edited zygotes. Genomic DNA analysis revealed disruptions of the mfviii gene reading frame through a 22-bp deletion and a 23-bp insertion in two separate founder mice. The founder mice showed all the clinical signs of hemophilia A including; excessive bleeding after injuries, and spontaneous bleeding in joints and other organs. Coagulation test data showed that mFVIII coagulation activity was significantly diminished in the mFVIII knockout (FVIIIKO) mice compared to normal mice. The CRISPR/nickase system was successfully applied to generate mouse lines with the knockout fviii gene. The two novel FVIIIKO mice demonstrated all clinical symptoms of hemophilia A, which could be successfully inherited. Therefore, both of the developed FVIIIKO mouse lines are eligible for being considered as proper mouse models of hemophilia A for in vivo therapeutic studies.

Optimizing the Expression and Solubilization of an E. coli-Produced Leukemia Inhibitory Factor for Anti-LIF Antibody Production and Use Thereof for Contraception in Mice.

Leukemia inhibitory factor (LIF) is an essential cytokine for blastocyst implantation. This study evaluated the effect of LIF inhibition on the blockage of embryo implantation. A truncated mouse LIF (tmLIF) was designed and expressed in E. coli. The protein expression was optimized using different culture media and inducers. To block pregnancy, the mice were immunized by the purified protein via maternal injection of the protein or in utero injection of the anti-LIF serum. The expression of implantation-relevant genes was quantified in the uterine tissue. The results showed that the protein was expressed in aggregated form in E. coli. The highest yield of protein was produced in the M9 medium. The insoluble protein was completely dissociated by SDS and 2-ME combination, but not by urea. The maternal immunization reduced the number of offspring, but not significantly. Instead, in utero injection of the anti-LIF serum prevented the blastocyst implantation. Gene expression analyses showed decrease of Jam2, Msx1and HB-EGF genes and increase of Muc1 gene as the result of intrauterine administration of the anti-LIF serums. In conclusion, SDS-mediated solubilization of inclusion bodies was compatible with in vivo studies. The intrauterine administration of anti-LIF serum could prevent mouse pregnancy. This indicates that in utero application of LIF antibodies might be used as a contraceptive.

Metformin modulates oncogenic expression of HOTAIR gene via promoter methylation and reverses epithelial-mesenchymal transition in MDA-MB-231 cells.

Epithelial-mesenchymal transition (EMT) is a biological event, which critically regulates migration and invasion of cancer cells. EMT is regulated by several protein and nonprotein factors (such as noncoding RNAs). HOTAIR is an oncogenic long noncoding RNA that stimulates EMT in cancers. In the current study, we investigated the effect of metformin on EMT behavior and HOTAIR expression in MDA-MB-231 breast cancer cells. The minimal effective concentrations of metformin (10 and 20 mM) were obtained by the MTT test. Cell migration and invasion in the metformin-containing medium were assayed in the scratch assay and transwell test. Meaningful decreases in both cell migration and invasion were observed in the presence of metformin. Vimentin, snail, ß-catenin, and HOTAIR transcripts were quantified by real-time polymerase chain reaction (PCR). Reduction in the expression of vimentin, ß-catenin, and HOTAIR was detected as the result of metformin treatment, but the snail showed a constant expression. Western blottingrevealed the downregulation of vimentin and ß-catenin proteins. HOTAIR promoter methylation pattern was also investigated in metformin-exposed cells using bisulfite sequencing PCR which the result showed differences in the methylation profile of CpG islands between the treated and untreated cells. In conclusion, metformin modulated oncogenic expression of the HOTAIR gene in the MDA-MB-231 cells. This downregulation was associated with the modification of promoter methylation patterns. Since HOTAIR induces EMT in breast cancer, HOTAIR decline might be one of the mechanisms by which metformin reverses EMT.

Immunization against leukemia inhibitory factor and its receptor suppresses tumor formation of breast cancer initiating cells in BALB/c mouse.

Immunotherapy is a promising approach for specific targeting of cancer cells. Leukemia inhibitory factor (LIF) regulates several features of cancers and cancer stem cells (CSCs) through binding to LIF receptor (LIFR). In this study, we investigated the consensus of LIF and LIFR immunization on the growth of mouse mammary tumors. For this purpose, mouse LIF and LIFR were designed as truncated proteins, expressed in E. coli and then injected to mice as individual and mixed antigens. The results showed the production of neutralizing antibodies and secretion of interferon-γ and interleukin-2 in response to immunization. In continue, the immunized mice were subjected for tumor formation challenge by inoculation of the breast CSCs derived from MC4-L2 cells. Development of the breast tumors was observed in all the control mice, while the tumors appeared in 75% of animals in the LIF group. LIFR injection, individually or in combination with LIF, strongly inhibited the tumor growth to only 25% of the mice. Moreover, a delay in tumor appearance was observed in the immunized mice compared to the controls. Immunostaining of the tumor sections confirmed the expression of LIF and LIFR. In conclusion, LIF and LIFR might be effective targets for immunotherapy of the tumors that express these proteins.

Isolation and characterization of breast cancer stem cell-like phenotype by Oct4 promoter-mediated activity.

Cancer stem cells (CSCs) are a small subset of cancer cells responsible for self-renewal activity, drug resistance, and tumor recurrence. CSCs have been derived from diverse tumors and cell lines. The expression of stemness markers has been identified in CSCs. Oct4 is a well-established transcription factor expressed in stem cells and CSCs. In this study, we isolated and characterized breast CSC-like cells from murine MC4-L2 cells by Oct4 promoter-mediated activity. The MC4-L2 cells were electroporated by a plasmid expressing puromycin resistance (PuroR ) gene from the Oct4 promoter and then selected by puromycin. The isolated cells were named as the MC4-L2puro cells and characterized for CSCs properties. Immunostaining indicated CD44high and CD24high phenotype for the MC4-L2 and MC4-L2puro cells. The enhanced expression of stem cell markers was detected in the puromycin-selected cells compared with the parental cells. Moreover, the isolated cells only grew up in sphere-formed shape in low attachment plates. Serial dilution transplantation in syngeneic mouse models showed increased tumorigenicity of the MC4-L2puro cells, as they induced new tumors when injected into the mammary fat pad as few as 104 cells. In conclusion, we designed a novel genetic construct, which allows the isolation of Oct4-positive cells in a cancer population by a simple selection step in a puromycin-containing medium. Transfection of this construct into the MC4-L2 cells resulted in growing a subpopulation of cells having tumor-initiating cell characteristics. To the best of our knowledge, this is the first report on the isolation of CSC-like cells from the mouse breast cancer MC4-L2 cells.

Preparation of brushite cements with improved properties by adding graphene oxide.

Background: Brushite (dicalcium phosphate dihydrate, DCPD) cement as a promising bioactive material for bone tissue engineering is widely used to treat defects. However, relatively poor mechanical properties of brushite cement limit its application in loadbearing conditions. The aim of this study is to investigate the effect of graphene oxide (GO) addition to the physical-mechanical-biological properties of brushite cement. Methods: The brushite types of cement were prepared by mixing ß-tricalcium phosphate [ß-TCP, Ca3 (PO4)2] and monocalcium phosphate monohydrate [MCPM, Ca(H2PO4)2. H2O]. GO was introduced at 0, 0.5, 2, and 5 wt.% with the liquid. MG63 cells were cultured on the GO/CPC surfaces to observe various cellular activities and hydroxyapatite (HA) mineralization. Results: Based on our results, GO/CPC composites exhibit improvement in compressive strength compared to pure CPC. New Ca-deficient apatite layer was deposited on the composite surface after immersing immersion in SBF for 7 and 14 days. Field emission scanning electron microscope (FESEM) images indicated that pure and GO incorporated brushite cement facilitates cell adhesion. CPC/GO was slightly toxic to cells such that high concentrations of GO decreased the cell viability. Besides, alkaline phosphatase (ALP) activity of cells was improved compared with the pure CPC. Conclusion: Our results highlight the role of graphene oxide that may have great potential in enabling the utility of graphene-based materials in various biomedical applications.

The chicken hypersensitive site-4 insulator cannot fully shield the murine phosphoglycerate kinase-1 promoter from integration site effects in transgenic mice.

Differential expression of transgenes in transgenic animals is one of the main drawbacks of pronuclear injection. To overwhelm this issue, the genetic constructs are equipped with insulators. In this study, the consensus of exerting chicken hypersensitive site-4 (cHS4) insulator was examined on the shield of phosphoglycerate kinase-1 (Pgk-1) promoter from the surrounding chromatin in transgenic mice. The PGK-EGFP cassette was flanked by insertion of three copies of the cHS4 insulators. Mouse zygotes' microinjection by the constructed cassette was resulted in the birth of nine transgenic founders (F0). Copy-number-dependent expression of the EGFP was investigated in the transgenic F1 offspring by fluorometry and real-time PCR. They showed no correlation between the expression level of transgene and gene copy number among the transgenic lines. Moreover, dissection of the EGFP-expressing mice revealed heterogeneous expression of the EGFP in the different organs. In conclusion, for the first time, these findings indicated that the cHS4 sequence is not a perfect insulator to fully protect the Pgk-1 promoter from the side effects of integration site in transgenic mice and it needs probably to some additional elements of the cHS4 locus to reach this goal.