ABSTRACT
The gene transfer efficiency into nonobese diabetic/severe combined immunodeficient (NOD/SCID)-repopulating cells (SRCs) derived from umbilical cord blood (UCB) (n = 11 NOD/SCID mice) and granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (MPB) (n = 64 NOD/SCID mice) was compared using a clinically relevant protocol and a retrovirus vector expressing the enhanced green fluorescent protein (EGFP). At 6-9 weeks after transplantation, the frequency of transduced human cells in the bone marrow (BM) (40.5% +/- 2.4% [mean +/- SE]) and spleen (SPL) (36.4% +/- 3.2%) in recipients of UCB cells was significantly higher (p < 0.001) than that observed in the BM (2.2% +/- 1.8%) and SPL (2.0% +/- 2.6%) in recipients of MPB. In subsequent studies, MPB was cultured for 2-8 days in cytokines prior to transduction to determine if longer prestimulation was required for optimal gene transfer. A significant increase in gene transfer into CD45(+) human cells and clonogenic cells derived from MPB SRCs was observed when cells were prestimulated for 6 days compared to 2 days prior to transduction (p = 0.019). However, even after 6 days of prestimulation, transduction was still significantly less than UCB. A substantial discrepancy exists in the ability to introduce genes effectively via retrovirus vectors into SRCs derived from MPB as compared to UCB.
Subject(s)
Blood Cells/drug effects , Blood Cells/metabolism , Blood Transfusion , Fetal Blood/metabolism , Granulocyte Colony-Stimulating Factor/pharmacology , Severe Combined Immunodeficiency/immunology , Transduction, Genetic/methods , Animals , Blood Cells/cytology , Blood Cells/transplantation , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Bone Marrow Transplantation , Colony-Forming Units Assay , Fetal Blood/cytology , Flow Cytometry , Gene Expression , Genetic Therapy/methods , Green Fluorescent Proteins , Humans , Leukocyte Common Antigens/analysis , Leukocyte Common Antigens/immunology , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Polymerase Chain Reaction , Retroviridae/genetics , Spleen/cytology , Spleen/metabolism , Time Factors , Transgenes/genetics , Transplantation ImmunologyABSTRACT
The objective of this study was to profile mRNA expression of argininosuccinate synthetase (AS) and ornithine transcarbamylase (OTC), two enzymes that participate in the formation of urea in liver and compare these with changes in mRNA for pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK) during the periparturient period in dairy cows. Forty-eight multiparous Holstein cows were fed isoenergetic prepartum diets that contained 10% RDP and either 4.0% RUP or 6.2% RUP and either 0, 6, or 12 g/d of rumen-protected choline (RPC) as CapShure (Balchem Corp., Slate Hill, NY). After calving cows received a common diet and continued RPC as per their prepartum assignments. Liver biopsies were obtained on d -28, -14, 1, 28, and 56 relative to calving, and the abundances of AS, OTC, PC, PEPCK, and 18S mRNA were determined by Northern blot analysis of total RNA. The abundance of OTC mRNA was lowest at calving and was decreased by RPC and 6.2% RUP feeding. Feeding 6.2% RUP did not alter AS, PC, or PEPCK mRNA. The expression of AS mRNA increased and PEPCK mRNA tended to increase from calving to 56 DIM. Pyruvate carboxylase mRNA increased more than twofold at calving. The data indicated adaptation to lactation for gluconeogenic enzymes that is not matched in direction and magnitude by changes in mRNA for urea cycle enzymes. Feeding additional protein, as RUP, failed to induce mRNA for key enzymes in gluconeogenesis or ureagenesis.
Subject(s)
Gluconeogenesis/genetics , Lactation/metabolism , Liver/enzymology , RNA, Messenger/genetics , Rumen/metabolism , Urea/metabolism , Animal Feed , Animals , Biopsy , Blotting, Northern , Cattle , Choline , DNA, Complementary , Dietary Proteins/administration & dosage , Female , Gene Expression Regulation, Enzymologic , Gluconeogenesis/physiology , Liver/metabolismABSTRACT
The objectives of the present study were to determine the effects of rumen undegradable protein (RUP) level of prepartum diets, the supplementation of a rumen-protected choline product, and their interactions on milk production, feed intake, body weight and condition, blood metabolites, and liver triacylglycerides in dairy cows. Rumen-protected choline (RPC) was fed with two levels of RUP to 48 multiparous Holstein cows in a 3 x 2 factorial arrangement of treatments. Beginning 28 d before expected calving, cows were fed 10% rumen degradable protein, either 0, 6, or 12 g/d of RPC as CapShure (Balchem Corp., Slate Hill, NY) and either 4.0 or 6.2% RUP. After calving and through 120 d of lactation, cows received a common diet and continued RPC as per their prepartum assignment. Prepartum dry matter intake (kg/d) was not affected by RPC or RUP. Postpartum intake decreased when 6.2% RUP was fed prepartum. Milk production to 56 d in milk was decreased when cows were fed 6.2% RUP prepartum. Milk protein (kg/d) decreased when additional RUP was fed prepartum. Cows fed RPC lost more weight during the study period and tended to lose more body condition. Plasma urea nitrogen levels in the prepartum period were reduced for cows fed 4.0% RUP prepartum. Mean liver triacylglyceride determined from samples obtained at -28, -14, +1, +28, and +56 d in milk was not affected by RPC, prepartum RUP, or their combinations. Feeding 12 g of RPC/d in conjunction with 4.0% RUP increased milk production, but feeding RPC with 6.2% RUP prepartum and through 56 d in milk decreased production. The data indicate that 6.2% RUP does not benefit close-up dry cows, and the response to RPC depends the RUP content of the prepartum diet.
