Detection of Antisense to IGF2R (AIR) RNA in Cattle

Antisense;Epigenetics;Bovine;Large Offspring Syndrome;Igf2r

Farmer, William Taylor Quinton ; Jorge A. Piedrahita, Committee Member,Charlotte E. Farin, Committee Chair,Peter W. Farin, Committee Co-Chair,Farmer, William Taylor Quinton ; Jorge A. Piedrahita ; Committee Member ; Charlotte E. Farin ; Committee Chair ; Peter W. Farin ; Committee Co-Chair

University:North Carolina State University

关键词: Antisense;    Epigenetics;    Bovine;    Large Offspring Syndrome;    Igf2r;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/2559/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

FARMER, WILLIAM TAYLOR QUINTON.Detection of Antisense to IGF2R (AIR) RNA in Cattle.(Under the direction of Charlotte Farin.)The insulin-like growth factor type 2 receptor (Igf2r) regulates fetal growth by removing Igf2 from circulation, thus preventing overgrowth. In mice, expression of the Igf2r gene is imprinted only after implantation and is associated with expression of the antisense non-coding (nc)RNA, Air. In contrast, the human IGF2R gene is not imprinted and AIR ncRNA does not exist. Because it is known that IGF2R is imprinted in cattle, the objectives of this study were to determine if AIR ncRNA exists in cattle;if so, whether bovine AIR (bAIR) expression changes at developmentally important stages of gestation, and whether method of embryo production affects air expression. For objective 1, primer sets were designed for bAIR based on bovine genomic sequence. The primer set, bAIR3, was used to amplify a region of bAIR corresponding to an antisense segment within intron 1 of IGF2R. Primer set bAIR4 amplified a segment of bAIR ncRNA corresponding to an antisense region upstream of the 5'-untranslated region of IGF2R. Whole cell RNA was extracted from liver samples of bovine fetuses at day 70 of gestation resulting from the transfer of either in vivo-produced (n=7, IVO) or in vitro-produced (n=6, IVP) embryos.Extracted RNA was subjected to DNase treatment, reverse transcription (RT) and PCR. Control RT reactions included RT without Superscript III (Invitrogen) and RT without Superscript III or DNase. Controls confirmed that amplification products resulted from RNA present in the sample and not from genomic DNA contamination. Amplicons were obtained for both the bAIR3 and bAIR4 primer sets and were sequence verified.These results demonstrated that bAIR ncRNA does exist in cattle. For objective 2, conceptuses (n=9, IVO, mean ± sem length: 2.2 ± 0.6mm) were recovered from cows at day 15 of gestation and snap-frozen for RNA extraction. Blastocysts (n=2 pools of 20 IVO embryos and n=4 pools of 25 to 27 IVP embryos) were recovered from cows on day 7 of development and snap frozen for RNA extraction.Semi-quantitative RT-PCR assays were performed to assess levels of IGF2R mRNA, H2A mRNA and bAIR ncRNA.Relative RNA expression was calculated as the ratio of band intensities of the RNA of interest to that of H2a.Data on levels of expression in fetal liver between IVO and IVP treatment groups were analyzed by Student’s T-test.H2A mRNA was expressed in all day 70 fetal liver samples, day 15 conceptuses, and day 7 blastocyst pools.IGF2R mRNA was expressed in all fetal liver samples, in 8 of 9 day 15 conceptuses, and in all day 7 blastocyst pools. bAIR ncRNA was expressed in 7 of 7 samples of day 70 fetal liver.In contrast, only 1 of 9 conceptuses expressed a bAIR ncRNA signal based on the bAIR3 primer set whereas 8 of 9 conceptuses expressed bAIR ncRNA based on the bAIR4 primer set.No bAIR ncRNA was expressed in any blastocyst pools based on either the bAIR3 or bAIR4 primer sets.Relative levels of bAIR ncRNA were greater (P<0.05) in fetal liver generated from the transfer of in vivo-produced embryos compared to that from in vitro-produced embryos (IVO: 0.426 ± 0.090 vs. IVP: 0.112 ± 0.098).In summary, the antisense ncRNA AIR exists in cattle and is expressed following implantation.Furthermore, the relative level of bAIR ncRNA can be altered by method of embryo production. These observations are consistent with data from the mouse and suggest that bAIR may be involved in regulating imprinted expression of IGF2R in cattle.Supported by the NC Agricultural Experiment Station and the NCSU College of Veterinary Medicine.