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.

Differential proteome analysis of a selected bacterial strain isolated from a high background radiation area in response to radium stress.

The present study describes the response of a bacterial strain, isolated from a hot spring in an area with the highest levels of natural radiation, under radium ((226)Ra) stress. The bacterium has been characterized as a novel and efficient radium biosorbent and identified as a variant of Serratia marcescens by biochemical tests and molecular recognition. In order to gain insights into key cellular events that allow this strain to survive and undergo (226)Ra adaptation and biosorption, the strain was tested under two experimental conditions of 1000 and 6000 Bq (226)Ra stress. A proteomic approach involving two-dimensional polyacrylamide gel electrophoresis and mass spectrometry was used to identify the differentially expressed proteins under (226)Ra stress. Functional assessment of identified proteins with significantly altered expression levels revealed several mechanisms thought to be involved in (226)Ra adaptation and conferring resistant phenotype to the isolate, including general stress adaptation, anti-oxidative stress, protein and nucleic acid synthesis, energy metabolism, efflux and transport proteins. It suggests that this strain through evolution is particularly well adapted to the high background radiation environment and could represent an alternative source to remove (226)Ra from such areas as well as industrial radionuclide polluted wastewaters.