Title page for ETD etd-04182012-144142

Type of Document Dissertation
Author Miller, Carin R
URN etd-04182012-144142
Degree PhD
Department Animal and Poultry Sciences
Advisory Committee
Advisor Name Title
Gerrard, David E. Committee Chair
Dalloul, Rami A. Committee Member
Huckle, William R. Committee Member
Jiang, Honglin Committee Member
Tu, Zhijian Jake Committee Member
  • Amino Acid
  • RNAi
  • Transgenic
  • Chicken
  • PepT1
  • Enterocyte
Date of Defense 2012-04-04
Availability unrestricted
Amino acids are assimilated by membrane-associated transporters into and out of enterocytes either in their free form or in the form of peptides. The peptide transporter, PepT1, is thought to be the major facilitator of peptide transport in the enterocyte. It is unknown if the peptide transporters and free amino acid transporters operate in a compensatory fashion to regulate the amino acid balance within the enterocyte. Therefore, the objective was to examine the regulatory balance between PepT1 and other peptide and free amino acid transporters in enterocytes.

The Mouse Small Intestinal Epithelial (MSIE) cells are conditionally immortalized. It was found that MSIE cells express BoAT1, CAT1, CAT2, LAT1, y+LAT1, and y+LAT2, but not PepT1, EAAT3, Bo,+AT, or LAT2, making this model similar to the basolateral membrane of enterocytes. Growing MSIE cells at high temperatures did not affect the nutrient transporter gene expression profile of these cells. Thus, the human colon carcinoma (Caco-2) cell line was used as a small intestinal in vitro model for this study. These cells express PepT1, HPT1, PTR3 EAAT1, EAAT3, rBAT, Bo,+AT CAT1, LAT1, y+LAT1, y+LAT2, ABCC3, ABCC4, which increased from D0 to D21 post confluency, indicating cell maturation. In Caco-2 cells, PepT1 gene silencing was induced in Caco-2 cells. Despite an reduction of PepT1 gene (82%, P < 0.05) protein (96%), no significant difference in any peptide (HPT1, PTR3, ABCC3, ABCC4) or free amino acid transporters (EAAT1, EAAT3, rBAT, Bo,+AT, BoAT1, CAT1, CAT2, LAT1, LAT2, y+LAT1, y+LAT2) between Caco-2 cells treated with PepT1 siRNA and Caco-2 cells treated with Control siRNA was observed. These results suggest no compensation at the gene expression level of these transporters in response to a reduction of PepT1.

To account for the limitations of an in vitro and PepT1 kockout mouse model, transgenic chicken models were pursued. Potential cPepT1 overexpressing, cPepT1 shRNA or control shRNA expressing G0 chickens were generated by embryo injection of pseudolentiviral particles followed by ex ovo egg culture. Overall, 9 potential G0 cPepT1 overexpressing chickens, 15 potential G0 cPepT1 shRNA expressing chickens, and 4 potential G0 control shRNA expressing chickens were generated.

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