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Title: Development of a transgenic goat model wih cardiac-specific overexpression of transforming growth factor - {beta} 1 to study the relationship between atrial fibrosis and atrial fibrillation

Author
item POLEJAEVA, I - Utah State University
item HALL, J - Utah State University
item MENG, Q - Utah State University
item ZHOU, X - Utah State University
item SESSIONS, B - Utah State University
item Panter, Kip
item STOTT, R - Utah State University
item RUTIGLIANO, HELOISA - Utah State University
item DAVIES, CHRISTOPHER - Utah State University
item WANG, ZHONGDE - Utah State University
item RANJAN, RAVI - University Of Utah
item DOSDALL, DEREK - University Of Utah
item MACLEOD, ROB - University Of Utah
item MARROUCHE, NASSIR - University Of Utah
item WHITE, KENNETH - Utah State University

Submitted to: Circulation Research
Publication Type: Abstract Only
Publication Acceptance Date: 9/15/2012
Publication Date: 10/1/2012
Citation: Polejaeva, I.A., Hall, J., Meng, Q., Zhou, X., Sessions, B.R., Panter, K.E., Stott, R., Rutigliano, H.M., Davies, C.J., Wang, Z., Ranjan, R., Dosdall, D., Macleod, R., Marrouche, N., White, K.L. 2012. Development of a transgenic goat model wih cardiac-specific overexpression of transforming growth factor - {beta} 1 to study the relationship between atrial fibrosis and atrial fibrillation. Circulation Research. 111: A251.

Interpretive Summary: Studies on patients, large animal models and transgenic mouse models have shown a strong association of atrial fibrosis with atrial fibrillation (AF). However, it is unclear whether there is a causal relationship between atrial fibrosis and AF or whether these events appear as a result of independent pathological changes in the heart. We are testing the hypothesis that goats that overexpress TGF-ß1 (transforming growth factor beta1) specifically in cardiac myocytes will develop atrial fibrosis that in turn will lead to AF. Many aspects of AF-related remodeling have been studied comprehensively in goat models. However, these AF models are typically mechanically induced (eg, the rapid atrial pacing model). This unique transgenic goat model has the potential to offer insights into the role of fibrosis in AF initiation and progression without the confounding effects of mechanical AF induction. Somatic cell nuclear transfer (SCNT or cloning) was used to produce TGF-ß1 transgenic pregnancies. First, pcDNA3.1DV5-MHC-TGF-ß1cys33ser vector was constructed by subcloning the MHC-TGF-ß1 fragment from the plasmid pUC-BM20-MHC-TGF-ß1 into the pcDNA3.1D V5 vector. The NeonTM transfection system was used to electroporate primary goat fetal fibroblasts. After two weeks of G418 selection, the resulting G418 resistant colonies were screened by PCR to confirm transgene integration into goat genomic DNA. PCR positive cells were used for SCNT. Cloned embryos (n=264) were cultured for 12-60 h in vitro and then transferred into synchronized recipient females (n=15). Confirmation of pregnancy was done by ultrasonography on day 30 post-transfer. At the time of this abstract submission, 40% (6/15) of recipients were confirmed to be pregnant as determined by the presence of a heartbeat. The range for the stage of gestation is between day-60 and day-120. The first delivery date is April 28, 2012. Several reports documented no pregnancy losses after 30 days of gestation in goats. Therefore, we expect that most if not all of these pregnancies will result in delivery of live offspring. To our knowledge, this will be the first transgenic goat model generated for cardiovascular research.

Technical Abstract: Studies on patients, large animal models and transgenic mouse models have shown a strong association of atrial fibrosis with atrial fibrillation (AF). However, it is unclear whether there is a causal relationship between atrial fibrosis and AF or whether these events appear as a result of independent pathological changes in the heart. We are testing the hypothesis that goats that overexpress TGF-ß1 (transforming growth factor beta1) specifically in cardiac myocytes will develop atrial fibrosis that in turn will lead to AF. Many aspects of AF-related remodeling have been studied comprehensively in goat models. However, these AF models are typically mechanically induced (eg, the rapid atrial pacing model). This unique transgenic goat model has the potential to offer insights into the role of fibrosis in AF initiation and progression without the confounding effects of mechanical AF induction. Somatic cell nuclear transfer (SCNT or cloning) was used to produce TGF-ß1 transgenic pregnancies. First, pcDNA3.1DV5-MHC-TGF-ß1cys33ser vector was constructed by subcloning the MHC-TGF-ß1 fragment from the plasmid pUC-BM20-MHC-TGF-ß1 into the pcDNA3.1D V5 vector. The NeonTM transfection system was used to electroporate primary goat fetal fibroblasts. After two weeks of G418 selection, the resulting G418 resistant colonies were screened by PCR to confirm transgene integration into goat genomic DNA. PCR positive cells were used for SCNT. Cloned embryos (n=264) were cultured for 12-60 h in vitro and then transferred into synchronized recipient females (n=15). Confirmation of pregnancy was done by ultrasonography on day 30 post-transfer. At the time of this abstract submission, 40% (6/15) of recipients were confirmed to be pregnant as determined by the presence of a heartbeat. The range for the stage of gestation is between day-60 and day-120. The first delivery date is April 28, 2012. Several reports documented no pregnancy losses after 30 days of gestation in goats. Therefore, we expect that most if not all of these pregnancies will result in delivery of live offspring. To our knowledge, this will be the first transgenic goat model generated for cardiovascular research.