[Expression of multiple cancer/ testis genes in 32 breast cancer tumor samples]

Cancer/testis genes [CT-genes] are a family gene which only express in normal testis tissue; some of them are randomly expressed in some types of cancers. The aim of this study was to evaluate the frequency of expression of CT-genes in the patients with breast cancer. Thirty two breast cancer tissue samples were prepared. Expression of NY-ESO-1 1a, NY-ESO-1 1b, SCP1, SSX-2 and MAGE-3 genes, as well as GAPDH [internal control], were studied by multiplex RT-PCR method. Three [9%] of 32 tumor samples expressed mRNA of NY-ESO-1 1a, while six [19%] of 32 tumor samples expressed mRNA of NY-ESO-1 1b. Seven [22%] and two [6%] of 32 tumor samples expressed mRNA of SCP1 and mRNA of MAGE3, respectively. Overall, Thirteen [41%] samples expressed one of the studied genes. NY-ESO-1 and SCP1 genes had the highest frequency of expression of mRNA. It is suggested that more number of breast cancer tumor samples should be examined to evaluate expression of CT-genes. SCP1 and NY-ESO-1 proteins may promote future breast cancer immunotherapy

[ study of intron 22 inversion type I and II of coagulation factor 8 gene in patients with severe Hemophilia A using IS-PCR technique]

Hemophilia A [HA] is an X-linked recessive bleeding disorder. The disease is caused by mutations in the F8 gene. Inversion of intron 22 is the most common causative mutation of severe hemophilia A, which is detectable by southern blot and polymerase chain reaction [PCR] methods. The aim of this study was to determine intron 22 inversion type I and type II inversion in severe hemophilia A patients by inverse shifting-PCR method. This study was performed on 30 patients with severe hemophilia A with less than 1% of normal level activity of Factor 8. After obtaining consent from the patients, genomic DNA was extracted from peripheral blood leukocytes. The extracted DNAs were used as template for IS-PCR amplification after circularization [digestion by BcII and then ligation of the produced fragments by ligase enzyme]. In 30 severe hemophilia A patients, 40% of the patients had intron 22 inversion type I, and 6.6% of the patients had intron 22 inversion type II. The results of this study revealed that recombination between intron 22h-1 within the F8 gene and its copies [22h-2 and 22h-3], which lie in opposite direction to intron 22h-1, respectively cause the inversions of intron 22 type II and type I. Inversion of intron 22 type I is more frequent than type II. Also, by application of IS-PCR as a cost effective method, we could save time and improve the molecular detection of inversion. This method and can be used for detection of carriers and patients and for prenatal diagnosis of hemophilia A disease

Investigation of genetic association between PRODH gene and schizophrenia in Iranian population

Schizophrenia is a complicated, debilitative mental disorder. Evidence is emerging for the association of polymorphisms in PRODH gene and increased risk of schizophrenia. In the present research, we investigated relationship between of this gene and schizophrenia disease by means of a gene polymorphism using PCR-RFLP technique.150 persons suffering from acute Schizophrenia and 160 healthy persons volunteering for this project were bled. Based on intended SNP, pair of primers was designed by Oligo7 program and polymerase chain reaction [PCR] was performed by thermo cycler. Then the resulted reactive mixture was exposed to a special enzyme, which we had intended for our study. Finally, the fragments of enzyme cut were transferred on the gel [4%] and migration pattern of resulted components were compared in healthy and patient subjects, whereby obtaining genotypes of different persons in polymorphic position. We utilized SPSS 16.0 program for statistical investigation of the work and studied SNP 1945T>C and its relation with the disease in statistical population. Our findings showed a meaningful relation between the occurrence of this nucleotide mutation and its frequency in patients [given P value=0.00]. Results of this work indicate that PRODH gene can be considered to be a significant candidate in our population as a factor influencing the occurrence of Schizophrenia

[Study of the association between estrogen receptor alpha gene expression in the levels of RNA and protein in primary breast tumors]

Estrogen receptor alpha protein status is determined by routine immunohistochemistry analysis in all malignant breast tumors. This assay has its limitations. RNA based techniques are potential complements for immunohistochemistry but it must be noticed that gene silencing may occur at different levels from RNA to protein. The aim of this study was the comparison of the results from these two assays and characterizing the tumors subgroup in which gene expression occurs at RNA level but the target protein is absent. 92 primary breast tumors including their clinical and IHC results were collected before treatment. Estrogen receptor gene expression of tumors was studied by Reverse Transcription Polymerase Chain Reaction [RT PCR]. In this assay, GAPDH was used as a reference gene. 36.6% of tumors with negative estrogen receptor protein showed gene expression at mRNA level. In this subgroup most of the patient were older than 50 years and in stages 3 or 4 of breast cancer and had poor prognosis according to Nottingham prognostic index. Most cases of the perineural invasion have been seen in this subgroup. It seems that RT-PCR assay would enable us to recognize a subgroup of breast tumors with poor prognosis which expresses RNA but not protein

[Production of camel polyclonal antibody against vascular endothelial growth factor receptor-2 and performance evaluation in ELISA test]

In molecular approach, serum of camel contains a unique type of antibodies devoid of light chains since the light chain is missing, the heavy-chain antibodies should bind their antigen by one single domain, the variable domain of the heavy immunoglobulin chain. Vascular endothelial growth factor receptor-2 [VEGFR-2] is one of the important proteins in angiogenesis which over-expressed in many types of tumors. Two male dromedaries [Camelus dromedarius] were immunized against VEGFR-2. ELISA was used to evaluate the immunization process. Camel immune sera were fractionated with protein A and G affinity chromatography. Heavy chain antibodies tested for its specific reactivity in cell-based ELISA in recognition of VEGFR-2 on the cell surface of three cell lines; 293/KDR, HUVECs and A431. In ELISA test, the camel sera in 1/12800 dilution had the acceptable results. Furthermore, cell-based ELISA demonstrated that the polyclonal heavy chain antibodies bind to VEGFR-2 in 293/KDR and HUVECs cell lines. Single chain polyclonal antibodies against VEGFR-2 can be used for detection of soluble form of this protein by ELISA, and also flowcytometry, western blotting and immunohistochemistry

Transduction of COS-7 and K562 cell lines by recombinant lentiviral particles carrying miniLCR and B-globin gene: Introduction to gene therapy of major B-thalassemia

Beta-thalassemia is caused by absence of reduction of beta-globin chain synthesis. One of the effective therapeutic methods for this disease can be gene therapy by viral vectors. The capacity of lentiviral vectors is approximately 8 kb, we designed a 6 kb construct containing mini LCR and beta-globin gene instead of LCR region. The aim of this study is to make a recombinant lentiviruses containing miniLCR and beta-globin gene for transfer to the target cells for gene therapy of beta-thalassemia. HS2, HS3, HS4 segments [miniLCR] and beta-globin gene with 5' and 3' UTR were amplified from the genomic DNA of a normal individual by PCR. Each segment was cloned in pTZ57R/T vector and then sub cloned first into the pBGGT vector and finally into the pLenti-Dest vector. Final transfer vector and the three helper packaging plasmids [Plp1, Plp2 Plp/VSVG] were contransfected into 293T packaging cells using lipofectamine 2000. Harvested viruses were confirmed by RT-PCR on extracted RNA of these recombinant lentiviruses. The titer of lentiviral stock determined in a K562 cell line and compared with COS-7 cell line. The titer in both cell lines was the same. Optimum MOI for COS-7 cell line was 5 and when polybrene was used transduction increased by 2 fold. The remaining transduced COS-7 colonies were expanded and DNA was extracted. By PCR, random intergration construct into the genome was evaluated. The produced lentiviruses can be an appropriate means for effective transfer of the designed construct into dividing and non-dividing cells such as hematopoetic stem cells for transplantation of beta thalassemia patients. Efficiency of transduction by leniviruses is more than the gene targeting technique. Also units of HS2, HS3 and HS4 regions in mini LCR and selection of larger HS3 unit may increase the expression of beta globin gene