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CaMV 35S Promoter in GM Feed That Sickened Rats Transferred into Rat Blood, Liver, and Brain Cells

Researchers led by Hanaa Oraby at Egypt’s National Research Center in Cairo are not the first to look for horizontal transfer of genetically modified (GM) DNA into animal cells, but certainly among the first to do an experiment aimed at detecting it and succeeded [1]. Horizontal gene transfer is the direct uptake of DNA (or RNA) into cells and integration of the sequence into the cell’s genome. Some of us regard horizontal gene transfer as the most serious hidden hazard of genetically modified organisms (GMOs) released into the environment ([2] Horizontal Gene Transfer - The Hidden Hazards of Genetic Engineering (ISIS special report). But a prevailing culture of denial by vested interests and regulators has obstructed proper investigation until very recently (see [3] Horizontal Transfer of GM DNA Widespread, SiS 64).

A GMO is an organism with synthetic foreign DNA gene sequences inserted into its genome in a laboratory process of artificial genetic modification that bypasses normal reproduction. Part of the foreign DNA is a control element called a promoter that is necessary for expressing the foreign genes. The most widely used is the cauliflower mosaic virus (CaMV) 35S promoter (which is what enables the virus to hijack the cell for making endless copies of the virus). The CaMV 35S promoter is now in more than 80 % of all GM plants [4], and is the first test for the presence of GMOs in unknown samples.

Probing for CaMV 35S promoter in the rat diet and in rat tissues

The Cairo researchers used three pairs of primers - specific short anchoring sequences that bind by specific base-pairing to the opposite ends of the DNA segment of interest – so as to amplify different segments from the CaMV 35S promoter with PCR (polymerase chain reaction). The amplified segments can then be isolated and detected on electrophoresis. The primers together amplify nearly 80 % of the entire promoter sequence. The experimental diet was an ordinary lab chow containing 60 % yellow maize and 34 % soybean, but unlabelled as to whether it is GM or not. The presence of GM material in the diet was ascertained using PCR assay with the three pairs of primers, which all gave the expected positive results, indicating that the diet contained GM material (up to a maximum of 94 %, if both the soybean and maize were completely GM).

The experimental animals consisted of 29 male Wistar albino rats immediately after weaning (age three to four weeks), which were divided into two main groups. One was fed on the lab chow containing GM ingredients for three months, and were further divided into three subgroups of 5, 5, and 7 animals, euthanized after 30, 60, and 90 days. The other control group was fed on a balanced non-GM diet for the same period and euthanized at the end of the experiment.

The lab chow diet used all through this experiment gave positive results when screened with primers for CaMV 35S promoter. The expected 195 bp (base pairs) amplified product was detected in all samples of the GM diet, but was absent in the control diet made up with non-GM material to match the nutritional content of the GM diet. As further confirmation, the PCR product obtained from the GM diet was sequenced, and shown to have 100 % identity with the CaMV 35S promoter at nucleotide coordinate 7190-7380 of the CaMV on sequence alignment analysis using the GenBank database. It also showed 100 % sequence identity with a number of binary vectors used for transferring genes in the lab that also contain the CaMV 35S promoter.

PCR analysis of the different tissues showed amplified sequence segments of the expected sizes for the three pairs of primers - 70, 88 and 195 bp - in some of the DNA samples of blood, liver and brain of rats fed the GM diet after 30, 60 and 90 days. None of the three primers gave amplification product in DNA samples of tissues from the controls fed the non-GM diet.

The 195 bp segment amplified from DNA samples of liver and brain in rats fed GM diet was subjected to DNA sequencing, and comparison with GenBank database revealed 100 % identity with the CaMV whole genome at the same nucleotide coordinates 7190-7384 for the 35S promoter. Furthermore, it also showed 100 % identity with the PCR segment amplified from the GM diet, and with the binary vectors segments that are 100 % identical to the PCR product from the GM diet.

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