Vance of genetic variations that lead to aberrant HAS1 splicing. However, splicing is a complex process and there are likely to be many combinations of genetic changes that can lead to aberrant splicing of HAS1. In the present studies, we have introduced deletions and mutations into HAS1 constructs to identify some of the regions that influence aberrant intronic splicing, comparing the splicing patterns obtained in transfectants with those we detect in patients with MM. We find that introduced genetic variations in HAS1 constructs convert in vitro splicing patterns to the patterns seen in vivo in patients. We also find that genomic DNA from MM patients harbors novel recurrent mutations in HAS1 introns that appear to regulate aberrant splicing in transfectants. Our work suggests that aberrant intronic HAS1 splicing in MM patients relies on intronic HAS1 mutations that are frequent in MM patients but absent from healthy donors.selected DNA fragments. For constructs carrying G1?8 m, G19?28 m, G19?4 m, G25?8 m or G27?8 m, DNA subfragments were amplified either from the parental construct or from the G1?28 m derivative, then joined together by overlap CB5083 extension PCR. The accuracy of each construct was validated by DNA sequencing.Transient Expression and HAS1 Splicing AnalysisHeLa were originally obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were grown at 37uC, 5 CO2 in DMEM (I-BRD9 web Invitrogen) supplemented with 10 fetal bovine serum (Invitrogen). Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours post-transfection, cells were washed twice with PBS and total RNA was prepared by Trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was reversed transcribed using dT15 and Superscript II (Invitrogen). PCR was run for 30 cycles at 94uC for 30 s, 60uC for 30 s, and 72uC for 30 s using Platinum Taq DNA polymerase (Invitrogen). PCR products were analyzed by agarose gel electrophoresis or DNA fragment analysis. For DNA fragment analysis, fluorescence primer was used and products were mixed with size standard in formamide and analyzed on an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems). Data analysis was performed using GeneMapper software version 4.0. Primer sequences are summarized in Table 1.Materials and Methods Ethics StatementThe study was approved by the Ethics Committee of the Cross Cancer Institute and University of Alberta. Written informed consent was provided in accordance with the Declaration of Helsinki.Analysis of HAS1Vb/Vd Expression in Peripheral Blood Mononuclear Cells (PBMC)Peripheral blood samples were collected from normal individuals and MM patients at diagnosis. MM was identified based on consensus criteria. Normal blood was obtained from University of Alberta Hospital emergency room as anonymous samples from 102 individuals selected as being over the age of 50 and without any obvious hematological issues. PBMC were isolated by step gradient centrifugation (FicollPaque Plus; GE Healthcare). RT-PCR followed Transient expression and HAS1 splicing analysis section, except that amplification was run for 35 cycles using E3/E5I4 primer set (Table 1) and PCR products were analyzed by DNA fragment analysis.Plasmid ConstructionHAS1FL (FLc) and HAS1g345 (G345) have been previously described [20,21]. In brief, FLc is generated by cloning of HAS1 cDNA fragment into a mammalian expression vector pcDNA3 (Invi.Vance of genetic variations that lead to aberrant HAS1 splicing. However, splicing is a complex process and there are likely to be many combinations of genetic changes that can lead to aberrant splicing of HAS1. In the present studies, we have introduced deletions and mutations into HAS1 constructs to identify some of the regions that influence aberrant intronic splicing, comparing the splicing patterns obtained in transfectants with those we detect in patients with MM. We find that introduced genetic variations in HAS1 constructs convert in vitro splicing patterns to the patterns seen in vivo in patients. We also find that genomic DNA from MM patients harbors novel recurrent mutations in HAS1 introns that appear to regulate aberrant splicing in transfectants. Our work suggests that aberrant intronic HAS1 splicing in MM patients relies on intronic HAS1 mutations that are frequent in MM patients but absent from healthy donors.selected DNA fragments. For constructs carrying G1?8 m, G19?28 m, G19?4 m, G25?8 m or G27?8 m, DNA subfragments were amplified either from the parental construct or from the G1?28 m derivative, then joined together by overlap extension PCR. The accuracy of each construct was validated by DNA sequencing.Transient Expression and HAS1 Splicing AnalysisHeLa were originally obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were grown at 37uC, 5 CO2 in DMEM (Invitrogen) supplemented with 10 fetal bovine serum (Invitrogen). Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours post-transfection, cells were washed twice with PBS and total RNA was prepared by Trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was reversed transcribed using dT15 and Superscript II (Invitrogen). PCR was run for 30 cycles at 94uC for 30 s, 60uC for 30 s, and 72uC for 30 s using Platinum Taq DNA polymerase (Invitrogen). PCR products were analyzed by agarose gel electrophoresis or DNA fragment analysis. For DNA fragment analysis, fluorescence primer was used and products were mixed with size standard in formamide and analyzed on an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems). Data analysis was performed using GeneMapper software version 4.0. Primer sequences are summarized in Table 1.Materials and Methods Ethics StatementThe study was approved by the Ethics Committee of the Cross Cancer Institute and University of Alberta. Written informed consent was provided in accordance with the Declaration of Helsinki.Analysis of HAS1Vb/Vd Expression in Peripheral Blood Mononuclear Cells (PBMC)Peripheral blood samples were collected from normal individuals and MM patients at diagnosis. MM was identified based on consensus criteria. Normal blood was obtained from University of Alberta Hospital emergency room as anonymous samples from 102 individuals selected as being over the age of 50 and without any obvious hematological issues. PBMC were isolated by step gradient centrifugation (FicollPaque Plus; GE Healthcare). RT-PCR followed Transient expression and HAS1 splicing analysis section, except that amplification was run for 35 cycles using E3/E5I4 primer set (Table 1) and PCR products were analyzed by DNA fragment analysis.Plasmid ConstructionHAS1FL (FLc) and HAS1g345 (G345) have been previously described [20,21]. In brief, FLc is generated by cloning of HAS1 cDNA fragment into a mammalian expression vector pcDNA3 (Invi.
