Dr. Mae-Wan Ho and Prof. Joe Cummins go behind the smokescreen to expose the project which would perpetuate the intensive animal husbandry that created mad cow disease in the first place and is far from safe or ethical in terms of animal welfare
Scientists announced the successful creation of cloned cows that would not get infected by BSE (Bovine Spongiform Encephalitis) , the dreaded mad cow disease that devastated the beef industry in Britain more than 20 years ago  ( The Inside Story of BSE , SiS 32). In potential re-enactment of the BSE fiasco, government regulators, in this case, the United States Food and Drug Administration (FDA), is about to approve cloned meat and milk for sale, pronouncing them safe despite massive public opposition on both safety and ethical grounds  ( Is FDA Promoting or Regulating Cloned Meat and Milk? this series). Meanwhile, Codex Alimentarius, the United Nations' food standards agency, has put out a public consultation on transgenic food animals that are likely to be contaminated with dangerous vaccines, drugs and nucleic acids  ( GM Food Animals Coming , SiS 32). Is this a coincidence or a well-orchestrated regulatory road show to smooth the passage of cloned transgenic animals into the world market?
Cloning by somatic cell nuclear transfer (SCNT) is the key to propagating genetically modified (GM) animals, as they do not breed true, the transgene is either silenced or physically lost in subsequent generations. Dolly the cloned sheep was the first mammal to be created by SCNT, and although not herself transgenic, she was followed by an entire herd of cloned transgenic sheep producing human alpha-1 antitrypsin in milk. Unfortunately, Dolly had to be put down in 2002 at age six, and the transgenic herd destroyed a year later; the process proved neither technically efficient nor financially viable . The scientists involved, including Ian Wilmut the creator of Dolly, abandoned cloning livestock to concentrate their efforts into using SCNT to make human embryonic stem cells for research and tissue replacement. But others have obviously not given up.
The cloned cows that would not get infected by BSE was created by precise ‘gene targeting', which on the face of it, looked far more advanced than the conventional genetic modification that is highly unpredictable and uncontrollable, resulting in a great deal of mutations, DNA scrambling and other collateral damage to the host genome  ( FAQ on Genetic Engineering , ISIS tutorial) . Instead, gene targeting involves, in theory at least, a precise ‘knockout' of the gene coding for the offending prion protein responsible for BSE. And that may become the selling point for both cloned and transgenic animals.
Prion proteins occur normally in a harmless form. But by folding into an aberrant shape, the normal prion protein turns into a rogue, infectious agent that is able to convert other normal prion protein molecules to fold into the same aberrant shape [6-8] ( Living Test for Mad Cow Disease , SiS 28). Prions are thought to be responsible for a number of degenerative brain diseases, including scrapie (a fatal disease of sheep and goats), BSE in cows, a chronic wasting disease in deer and elk, Creutzfeldt-Jakob disease (CJD) and its variant, vCJD, fatal familial insomnia, kuru (a slowly progressing fatal brain disease in Papua New Guinea), Gertsmann-Straeussler-Scheinker disease (an unusual form of hereditary dementia), and possibly some cases of Alzheimer's disease.
Normal prion protein is encoded by a gene in mammals, and is found throughout the body, even in healthy people and animals. However, the prion protein in infectious material has a different structure and is resistant to proteases (the enzymes in the body that break down proteins ) as well as to heat, radiation and formaldehye, treatments that would normally have killed viruses, bacteria and other disease agents.
Transgenic cloned mice with both copies of the prion gene disrupted (knockout) by homologous recombination showed no gross abnormalities, and neither produced prions nor harboured prion infection. However, deletions of the prion gene that extended into flanking genes caused the knockout mice to suffer ataxia (shaky, unsteady movement) and Purkinje cell loss in the adults . One study found that mice devoid of prion protein had cognitive deficits that could be rescued by reconstitution of prion genes in neurons .
Sheep cell lines with both copies of the prion protein gene knocked out were used to clone lambs by SCNT. The four lambs born live soon succumbed, three at birth and one after 12 days .
Gene knockout by homologous recombination has been used extensively for gene function analysis in mice, where it is accomplished using embryonic stem (ES) cells that can be made to develop into transgenic mice after genetic modification. In all other species, ES cells suitable for gene targeting are not available, and somatic cells have to be used. The genetically modified cells are then cloned into embryos by SCNT, and the embryos used to produce cloned offspring.
Gene targeting of somatic cells has the disadvantage that somatic cells have a short lifespan, which limits selection of properly targeted cell colonies and a low frequency of homologous recombination compared with embryonic stem cells.
To get the full consequence of a genetic knockout, both copies of the gene must be disrupted. In mice, heterozygous knockout founders are bred to produce a homozygous (both copies of the gene identical) inbred line. This takes a long time in species with long generation times, and the animals generally suffer from consequences of inbreeding, resulting in defective or otherwise weak animals.
Researchers in several companies – Gemini Science, California, USA, Kirin Brewery, Tokyo, Japan, and Hematech, South Dakota, USA – have devised a method for gene targeting somatic cells to create homozygous knockout calves .
The method involves targeting one copy of a gene by homologous recombination of somatic cells to produce heterozygous cell lines, then ‘rejuvenating' the cell lines by producing cloned foetuses by nuclear transfer, re-isolating cells lines from the foetuses, and targeting the second copy of the gene to produce homozygous (both gene copies identical) cell lines that can then be cloned into calves. This process can be repeated indefinitely, so it is possible to produce cell lines and clones with multiple genes modified. It involves a lot of work for cloning laboratories as well as for surrogate dams to carry the foetuses and calves, but it does speeds up the creation of transgenic clones enormously.
In a first experiment with cows, the immunoglobulin m (IGHM) gene was targeted. The first step was to identify a polymorphic (existing in more than one form) gene marker next to the targeted gene that is different in the two chromosomes, so the two copies of the gene can be distinguished. A suitable vector was constructed that has fragments of the gene to be knocked out followed by two selection marker genes to select for recombinants. A puromycin antibiotic resistance gene, puro , flanked by loxP sites (see later) allows all cells that have taken up the vector to be selected in the presence of puromycin, followed by a negative selection marker, a diphtheria toxin A gene that will kill the cell in case of non-homologous recombination. Homologous recombinants will integrate the vector at the correct site, and only the puromycin resistance marker gene will be integrated, while the diphtheria toxin A gene is lost. If the vector integrates at the wrong site through non-homologous recombination, then both marker genes will be integrated and the diphtheria toxin gene will become expressed and kill the cell, at least in theory . However, non-homologous recombinants also survived the diphtheria toxin selection, about half of them in fact, as described in the original paper in 1993, though this is not mentioned again in later publications.
The vector is introduced into cells by means of electroporation, literally, making holes in the cell membrane with a strong transient electric field, so the foreign DNA goes through into the cell.
The ‘targeting' is by no means precise, and it is difficult to tell how much it improves on conventional genetic modification. Of 446 wells that contained cells resistant to puromycin, only two (0.45 percent) were correctly targeted, which meant that more than 95 percent of the cells contained the transgenic vector integrated elsewhere in the genome, even with the negative selection against non-homologous recombinants. It is also very likely that the 0.45 percent of the cells ‘correctly targeted' may contain vector sequences integrated elsewhere in the genome, and that these non-homologous integrations contain the lethal diphtheria toxin A gene, as this possibility has not been ruled out.
The ‘correctly targeted' cells were then used for embryonic cloning to generate foetuses and rejuvenate the cell lines. This involved 30 embryos implanted in 15 cows, and a pregnancy rate of 50 percent. At 60 days gestation, six foetuses were collected, three of them confirmed to have one copy of the targeted gene, two in the A chromosome and one in the B chromosome, and were used to establish three cell lines. Cloned embryos were produced from all three cell-lines and transferred to 153 recipients to produce 13 (8 percent) “healthy” calves.
To target the second copy of the gene, a second knockout vector was constructed with a neo gene for neomycin resistance driven by an ST promoter (SV40 promoter and thymidine kinase enhancer). SV40 is a virus associated with malignant human tumours , and introducing an enhanced promoter from the virus into cattle intended for food use is far from safe. Of the cell lines that already had the gene in the A chromosome knocked out, only 2 out of 1 211 wells (0.17 percent) resistant to the antibiotic had correctly targeted the second (B) copy of the gene. In the cell line with the B copy knocked out, 6 out of 569 wells (1.1 percent) had corrected targeted the A copy.
Selected homozygous cells were cloned into embryos to generate foetuses to rejuvenate the cell lines. Overall pregnancy rate was 45 percent (40 of 89). At 45 days of gestation, 5 foetuses from one well and 15 from another were evaluated. All 5 from the first and only 3 from the second contained correctly targeted cells, as shown by PCR analysis. Cloned embryos from five double knock out cell lines were created and transferred to surrogate dams for development to term. This resulted in 8 calves (6 percent) born and confirmed to be double knockouts by PCR and sequence analysis.
No analyses were done to show that the calves were free from non-homologous transgene integration and diphtheria toxin A gene. There is also the problem that the calves contain two antibiotic resistance genes.
In order to remove the two antibiotic resistance marker genes used in the sequential gene knockout, the Cre-loxP site-specific recombination system was used, which was why the loxP sites were included flanking the antibiotic resistance genes. The idea was that by introducing the Cre recombinase into the cells, it could recognize and bind to the loxP sites, and snip out the antibiotic resistance genes in between.
The double-knockout cells were transfected with a Cre recombinase expression plasmid so that the Cre recombinase expressed could cut out the antibiotic resistance genes without the plasmid being integrated into the genome. Multiple wells showed evidence of excision of both antibiotic resistance genes, and one was selected for foetal cloning and regeneration of cell lines. Pregnancy rate was 35 percent at 40-50 days (21 of 60). Five foetuses were recovered, all of which had both antibiotic markers removed; but all except one had the Cre recombinase plasmid integrated into the genome, which was not intended. Cre recombinase integrated into the genome is bad news, as it has the potential to scramble up the host genome (see below).
The sequential gene knockout procedure was applied to the prion protein gene, PRNP . The cell line that had double knockout IGHM in which both antibiotic resistance markers were removed, without the Cre recombinase expression-plasmid integrated, was used as starting material . The first copy of PRNP gene was knocked out with a vector containing the neo selection marker driven by the ST promoter, flanked by lox P sites. Of 203 antibiotic resistant wells, 13 (5.9 percent) had correctly targeted one of the PRNP gene. This was a considerably higher rate than for IGHM , which is not expressed; PRNP is actively transcribed, and actively transcribed genes tend to be more easily targeted.
Some wells were cloned to generate 28 pregnancies at 45 days of gestation (71 percent). Five foetuses were examined, all of which contained correctly targeted cells.
The second copy of PRNP was knocked out with a vector that has the neo gene replaced with puro . After selection, 17 (5.2 percent) of the wells contained targeted cells. Sequence analysis confirmed that the second copy of PRNP was correctly targeted, creating doubly homozygous knockout cells in 16 wells. Cells from the correctly targeted wells were cloned to produce foetuses. The pregnancy rate at 45 days was 68 percent, and 18 foetuses collected were confirmed to be correctly targeted.
With sequential gene targeting each targeting event required ~2.5 months from transfection to establishment of regenerated cell lines; therefore homozygous targeted calves could be created in 14 months (5 months for targeting both copies of the gene and 9 months of gestation), and doubly homozyogous targeted calves including Cre-mediated excision could be created in 21.5 months. In contrast, for cattle breeding a heterozygous founder to produce homozygous calves would require ~5 years and generation of double homozygotes from two heterozygous founders is impractical.
The researchers suggest using this sequential targeting strategy for complex genetic modifications in large animal species to produce cloned animals serving as models for human diseases, as biofactories for various therapeutic proteins, or spare organs and tissues for transplant into humans, and finally, for improving the efficiency of agricultural production; all highly questionable in terms of safety, ethics/animal welfare.
In the most recent publication , three cell lines with double knockout PRNP were produced by similar methods and used to clone embryos.
Of 85 embryos implanted, 14 live births were obtained, and 12 survived beyond 6 months of age. The report claims that detailed comparisons at 12 months of age revealed no significant differences from controls, and at 20 months, they were apparently healthy and normal in all respects compared with wild-type non-cloned cattle. They did not perform DNA, RNA, protein or metabolic profiling, which would have been more informative concerning the many epigenetic errors known to be introduced by SCNT cloning  and the genetic mutations and genome scrambling known to be introduced by genetic modification . There was no effect on brain development in two PRNP -/- cattle examined at 14 months (why only two?), and immune functions appeared intact in all respects. The PRNP -/- bulls reached sexual maturity at a normal age and semen was collected from two animals (why only two?) at 16 months of age. Sperms appeared normal and were capable of generating normal-looking blastocysts by in vitro fertilization. Twelve blastocysts were implanted and eight cows were pregnant at 40 d of gestation.
Two brain regions were collected from one 10 month-old PRNP -/- cattle for a protein misfolding cyclic amplification (PMCA) assay. As control for the assay, CNS tissues from the identical anatomical sites were obtained from an age, sex and breed matched wild-type, non-cloned calf. A brain homogenate from a BSE-infected cow was added to the assay mixture to start the misfolding reaction. No propagation of proteinase K-resistance misfolded BSE prion protein (PrP BSE ) was detected by western blot analysis (specific test for the protein) when PRNP -/- brain homogenates were used. In contrast, PrP BSE was readily amplified and detected when brain homogenates from the wild-type cattle were used as substrates. The PRNP -/- brain homogenates were also resistant to cattle infected with transmissible mink encephalopathy (TME).
The publication  claims that nine PRNP -/- cattle have remained healthy for at least 20 months after birth, and suggest that PRNP -/- cattle could be a “preferred source of a wide variety of bovine-derived products that have been extensively used in biotechnology.” Although a ban on feeding cattle rumen-derived meat-bone meal has greatly reduced BSE infections in cattle, the possibility cannot be completely excluded that some PrP BSE strains might have originated from “spontaneous” misfolding of the normal protein. So prionless cattle will also prevent BSE due to spontaneous misfolding.
One co-author, James Robl, chief scientific officer of Hematch of Connecticut, told the New Scientist in 2004 that the US company had only created the cell lines lacking the prion gene, and denied that cloned cows would be produced for food . He said that the aim of the work was to use BSE-free cows to produce pharmaceutical products such as human antibodies, but that these cows were unlikely to end up on the dinner plate. That turned out to be a smokescreen, as we point out, there is no guarantee that hazardous experimental cloned transgenic cows will not enter the human food chain if transgenic and cloned animals are approved for human consumption [3, 4].
The project raises the question of whether it is proper science to devote such Herculean efforts to creating prionless cows, as it would merely perpetuate an intensive, industrial animal husbandry regime that created BSE in the first place. According to the UK Soil Association, which certifies organic agricultural produce , “there has never been a recorded case of BSE in an animal that was born in an organic herd where full organic management was in place throughout the animal's life.” This claim could not be refuted by subsequent investigations carried out as part of the UK government's BSE enquiry.
Prionless cows are both transgenic and cloned, and hence subject to the potential hazards and also the questionable ethics of both the transgenic and the cloning processes, regardless of whether they are intended for our dinner plate or for producing pharmaceuticals. Cloning creates massive deaths and suffering for failed foetuses and calves and also for the numerous surrogate dams required, and transgenesis in combination with cloning increases the number of cloning steps, and hence multiplies deaths and suffering.
It is not clear whether the calves still carry antibiotic resistance markers with loxP sites, the enhanced promoter from the SV40 virus, the Cre recombinase, or indeed, the diphtheria toxin A gene integrated at non-target sites in the genome. All of these dangerous genes could be subject to horizontal gene transfer and recombination, and in the process, trigger cancer (if transferred to human cells in the case of the strong viral promoter), and create and recreate viruses and bacteria that cause disease epidemics . Cre-recombinase is known to scramble genomes (see Box 1).
In conclusion cloned transgenic animals should not be approved for commercial use, nor should public research funding go to support such projects. Cloned transgenic animals are far from safe on existing evidence, and certainly not ethical in terms of animal welfare.
Cre recombinase is known to produce chromosome damage in mammalian cells  both at loxP sites and cryptic lox sites.
The ‘site-specific' recombination Cre/ lox system was originally isolated from the bacteriophage (bacterial virus) P1. Cre recombinase catalyses recombination between two lox sites, splicing out any stretch of DNA in between.
The system was first used in plants to create sterile ‘terminator' crops in order to protect patented transgenic traits, but was later offered as a way of containing the spread of transgenes  (Terminator Technology in New Guises, i-sis news3 ). We predicted it would scramble genomes, as the site specificity was not absolute. This proved to be the case  (Terminator Recombinase Does Scramble Genomes, i-sis news7/8 ) .
The Cre/lox system was also extensively exploited in transgenic mice. Studies in the test-tube have shown that Cre recombinase can catalyse recombination between DNA sequences found naturally in yeast and mammalian genomes. These ‘illegitimate sites' often bear little sequence similarity to the lox element.
It has been shown that high levels of Cre expression in the sperm cells of heterozygous transgenic mice led to 100 percent sterility in the males, despite the absence of any lox sites in the transgenic mice genome .
Article first published 31/01/07
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