Aberrant Gene Expression in Organs of Bovine Clones: MATERIALS AND METHODS
The NT procedures have been described in detail by Gong et al.. The donor nuclei were obtained from skin fibroblast cells of elite Holstein cow (age, 4 yr) and from FF cells of a female fetus (gestational age, 40 days). Approximately 27.9% (n = 283) and 37.9% (n = 294) of reconstructed embryos derived from AF and from FF, respectively, developed into blastocysts, with 11 (14.9%, n = 74) and 10 (22.7%, n = 44) of the transferred embryos, respectively, developing into full-term calves. Six of the 11 calves from AF cells (ww, bw, cw, sw, yw, and lw) survived and remained healthy, and five (AF1-AF5) died. Six of 10 calves from the FF cells (dw, qq, tt, jm, xw, and aw) survived and remained healthy, and four (FF1-FF4) died. All of the deceased cloned cattle died within 48 h of birth.
The cloned animals were dissected immediately after death, and normal control calves produced by artificial insemination were killed within 48 h after birth. Samples of all major internal organs of deceased cloned calves and normal control calves were immediately collected and frozen in liquid nitrogen for later analysis. buy zyrtec online
Preparation of RNA
We extracted total RNA from organ samples of the deceased cloned calves and normal control calves using a TRIZOL RNA isolation kit (In-vitrogen). The RNA preparations were treated with RNase-free DNase I to remove possible contaminating DNA and were stored at —70°C.
Reverse transcription (RT) was done using an RT kit (Promega) with approximately 1 |xg of RNA in a total volume of 20 |xl. The RT reaction was conducted according to the manufacturer’s guidelines using oligo(dT) primers and AMV reverse transcriptase enzyme in a volume of 20 |xl to prime the RT reaction and produce cDNA. Tubes were heated to 65°C for 5 min to denature the secondary RNA structure. The RT reaction was completed by adding 5 U of Superscript RT enzyme (Promega) before incubation at 37°C for 1 h and then 70°C for 15 min.
Quantitative RT-Polymerase Chain Reaction
The quantification of all gene transcripts was carried out by real-time quantitative RT-polymerase chain reaction (PCR).
Gene expression levels were measured using a DNA Engine Opticon 2 fluorescence detection system (MJ Research) and DyNAmo SYBR Green qPCR kit (MJ Research). SYBR Green I was a double-stranded, DNA-specific fluorescent dye. The sequences of the PCR primers used for RT-PCR quantification are shown in Table 1. The PCR reaction mixture (20 |xl) contained 10 ^l of DyNAmo SYBR Green qPCR mix, 5 |xl of primer (0.3 |xM forward and 0.3 |xM reverse), and 5 |xl of cDNA template (<10 ng/|xl). The PCR protocol included uracil-W-glycosylase (UNG) enzyme incubation at 50°C for 2 min and an initial denaturation at 95°C for 10 min. This was followed by 40 cycles of 10 sec each at 94°C for DNA denaturation, 20 sec at different temperatures for annealing of primers, 20 sec at 72°C for primer extension, and 1 sec at a different elevated temperature for data acquisition. The annealing and data acquisition temperatures of primers are shown in Table 1. The reannealing step (at 72°C for 10 min) allowed reformation of fully duplexed DNA in performing the agarose gel analysis. The melting protocol called for heating from 65 to 95°C, holding for 1 sec at each temperature, with increases of 0.2°C per step All samples were measured in triplicate. Performing the data-acquisi-tion step at an elevated temperature was helpful in minimizing the interference of primer-dimers with the quantification. Fluorescent data were acquired during the data-acquisition step. The melting curve analysis was used to check the specificity of an amplified product. Amplified product underwent electrophoresis on agarose/ethidium bromide gels and was visualized under ultraviolet (UV) light. In addition, product identity was confirmed by sequencing. The PCR products were purified using DNA wizard cleanup kit (Promega) and directly sequenced by Taq cycle sequencing using DyeDeoxy terminators in an automated sequencer (ABI 377; Applied Biosystems).
Expression was quantified by the relative standard curve method. The quantification was normalized to an endogenous RNA control Glycerai-dehyde-3-phosphate dehydrogenase (Gapdh a housekeeping gene), and standard curves were plotted for each target and the endogenous genes. Each of the cDNA fragments of the target gene was purified using DNA wizard cleanup kit and cloned into plasmids for use as standards in quantifying gene expression level. A standard graph of the cycle threshold (CT) values obtained from serial dilutions (10-104 copies/well) of the plasmid was created. Fluorescence was acquired in each cycle to determine the CT or the fluorescence baseline at which fluorescence rose above background for each sample. The Opticon Monitor 2 software produced a best-fit fluorescence baseline and the standard graph. For each experimental sample, the amounts of mRNA of each target gene and Gapdh were determined from the CT plotted on the respective standard curves. The mean values of the replicate wells run for each sample were calculated; subsequently, the mean quantity of each target gene was divided by Gapdh to obtain a normalized value for each transcript.
Because the experimental materials were the tissues with many serious aberrations, another endogenous RNA control jl-actin was used to identify the research results of the relative expression levels of target genes normalized by Gapdh.
Data were analyzed using SPSS (version 10.0; SPSS, Inc., Chicago, IL). An independent sample f-test was used to analyze differences in mRNA expression assayed by quantitative RT-PCR between the clones and the normal controls or between the AF-derived animals and the FF-derived clones. Differences were considered to be statistically significant at P< 0.05.
These studies were performed at the University of China Agriculture (Beijing, China), and all procedures were in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.