Tegration loci and its context rather than integrant numbers. Further studies have been carried out 
to survey the integrant loci and study the association with transgene expression. Our results also inferred that the level of promoter methylation played 22948146 much more important role in controlling transgene expression than that of integrant number in lentivirus-mediated transgenic sheep. Since the first publication on generation of transgenic sheep by injection of lentivirus into oocytes in 2009 [21], no further studies have been reported so far. Hereby, we are the first time to comprehensively investigate the issues of transgenic integrant, expression and methylation in lentivirus-mediated transgenic sheep. Taken together, we demonstrated that lentiviral transgenesis by injection of recombinant lentivirus into perivitelline space of sheep zygote could achieve high transgenic efficiency and high rate of transgene expression. Furthernore, the lentiviral transgene was subjected to alteration of methylation status and the transgene expression was inversely correlative with promoter methylation, whereas has no association with integrant numbers in lentivirusmediatied transgenic sheep.AcknowledgmentsWe thank Dr. Zhanjun Hou for carefully inspection of the manuscript. We also thank the team of management of sheep breeding farm, Biao Li, Bing Han and Fan Yang.Author ContributionsConceived and designed the experiments: ML CL JH WL. Performed the experiments: CL LW TC YT. Analyzed the data: CL NZ SH. Contributed reagents/materials/analysis tools: XZ. Wrote the paper: ML CL WL.
Brugada syndrome (BrS) is characterized by ST-segment elevation in the right precordial leads (V1?V3) of the electrocardiogram (ECG) with an associate risk of 94-09-7 cardiac arrhythmia [1]. The mean age of BrS clinical appearance is around 40 years with a strong male preponderance [2,3]. The ECG signature of BrS is transient and can be unmasked by administration of sodium channel blockers such as ajmaline or flecainide [2,4]. There are internationally accepted criteria to establish a diagnosis of BrS [5]. The prevalence is estimated to be approximately 1/2500. Although numerous environmental factors influence BrS clinical and ECG expressivity, it is commonly accepted that it is a genetic disease with usually an SMER28 autosomal dominant pattern of inheritance [6,7]. Since 1998, it has been established that about 15?5 of BrS cases can be linked to mutations in SCN5A that encodes the alpha subunit of cardiac sodium channel Nav1.5 [8]. Several othergenes have been implied in BrS such as GPD1L, CACNA1C, CACNB2, SCN1B, KCNE3, SCN3B, KCNJ8 [9], CACNA2D1 [10], KCND3 [11] and MOG1 [12] (for a review see [13]). The transient receptor potential melastatin protein number 4 (TRPM4) is a calcium-activated nonselective cation channel, member of a large family of transient receptor potential genes [14]. TRPM4 has been recently implied in families with 
progressive cardiac conduction blocks [15,16,17]. In this study, we addressed the question whether BrS cases could be attributed to TRPM4 mutations since BrS is frequently associated with cardiac conduction anomalies. In a large cohort of 248 BrS cases with no SCN5A mutation, 11 TRPM4 mutations were found in 20 unrelated individuals. The electrophysiological and cellular expression consequences of 4 mutations were further studied. These findings suggest that TRMP4 mutations accounts for about 6 of BrS.TRPM4 Mutations in Brugada SyndromeMaterials and Methods.Tegration loci and its context rather than integrant numbers. Further studies have been carried out to survey the integrant loci and study the association with transgene expression. Our results also inferred that the level of promoter methylation played 22948146 much more important role in controlling transgene expression than that of integrant number in lentivirus-mediated transgenic sheep. Since the first publication on generation of transgenic sheep by injection of lentivirus into oocytes in 2009 [21], no further studies have been reported so far. Hereby, we are the first time to comprehensively investigate the issues of transgenic integrant, expression and methylation in lentivirus-mediated transgenic sheep. Taken together, we demonstrated that lentiviral transgenesis by injection of recombinant lentivirus into perivitelline space of sheep zygote could achieve high transgenic efficiency and high rate of transgene expression. Furthernore, the lentiviral transgene was subjected to alteration of methylation status and the transgene expression was inversely correlative with promoter methylation, whereas has no association with integrant numbers in lentivirusmediatied transgenic sheep.AcknowledgmentsWe thank Dr. Zhanjun Hou for carefully inspection of the manuscript. We also thank the team of management of sheep breeding farm, Biao Li, Bing Han and Fan Yang.Author ContributionsConceived and designed the experiments: ML CL JH WL. Performed the experiments: CL LW TC YT. Analyzed the data: CL NZ SH. Contributed reagents/materials/analysis tools: XZ. Wrote the paper: ML CL WL.
Brugada syndrome (BrS) is characterized by ST-segment elevation in the right precordial leads (V1?V3) of the electrocardiogram (ECG) with an associate risk of cardiac arrhythmia [1]. The mean age of BrS clinical appearance is around 40 years with a strong male preponderance [2,3]. The ECG signature of BrS is transient and can be unmasked by administration of sodium channel blockers such as ajmaline or flecainide [2,4]. There are internationally accepted criteria to establish a diagnosis of BrS [5]. The prevalence is estimated to be approximately 1/2500. Although numerous environmental factors influence BrS clinical and ECG expressivity, it is commonly accepted that it is a genetic disease with usually an autosomal dominant pattern of inheritance [6,7]. Since 1998, it has been established that about 15?5 of BrS cases can be linked to mutations in SCN5A that encodes the alpha subunit of cardiac sodium channel Nav1.5 [8]. Several othergenes have been implied in BrS such as GPD1L, CACNA1C, CACNB2, SCN1B, KCNE3, SCN3B, KCNJ8 [9], CACNA2D1 [10], KCND3 [11] and MOG1 [12] (for a review see [13]). The transient receptor potential melastatin protein number 4 (TRPM4) is a calcium-activated nonselective cation channel, member of a large family of transient receptor potential genes [14]. TRPM4 has been recently implied in families with progressive cardiac conduction blocks [15,16,17]. In this study, we addressed the question whether BrS cases could be attributed to TRPM4 mutations since BrS is frequently associated with cardiac conduction anomalies. In a large cohort of 248 BrS cases with no SCN5A mutation, 11 TRPM4 mutations were found in 20 unrelated individuals. The electrophysiological and cellular expression consequences of 4 mutations were further studied. These findings suggest that TRMP4 mutations accounts for about 6 of BrS.TRPM4 Mutations in Brugada SyndromeMaterials and Methods.
