[Date Prev][Date Next][Thread Prev][Thread Next][Date Index][Thread Index]
[SANET-MG] Cloned BSE-Free Cows, Not Safe Nor Proper Science
Cloned BSE-Free Cows, Not Safe Nor Proper Science
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
This article has been submitted to the FDA on behalf of ISIS. Please
circulate widely to your elected representatives
An orchestrated regulatory road show
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.
Precise gene targeting claimed
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.
Normal proteins that cause disease by misfolding
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.
Gene knockout in mice
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.
Sequential gene knockout and SCNT cloning in cattle
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
In a first experiment with cows, the immunoglobulin ? (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).
Knockout of prion protein gene in cattle
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
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
Prionless cows promoted for preventing BSE
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 (PrPBSE) was detected by
western blot analysis (specific test for the protein) when PRNP-/- brain
homogenates were used. In contrast, PrPBSE 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 PrPBSE strains might have originated from ?spontaneous?
misfolding of the normal protein. So prionless cattle will also prevent
BSE due to spontaneous misfolding.
Large questions remain over the propriety, safety, ethics of creating
cloned prionless cows
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.
The Cre-lox system scrambles genomes
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
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
1. Richt JA, Kasinathan P, Hamir AN, Castilla J, Sathiyaseelan T, Vargas
F, Sathiyaseelan J, Wu H, Matsushita H, Koster J, Kato S, Ishida I, Soto
C, Robl JM and Kuroiwa Y. Production of cattle lacking prion protein.
Nat Biotechnol. 2006 Dec 31; [Epub ahead of print] doi:10.1038/nbt1271
2. Saunders PT. The inside story of BSE. Science in Society 32, 8-9, 2006.
3. Ho MW and Cummins J. Is FDA regulating or promoting cloned meat and
milk? ISIS Report, January 20.
4. Cummins J and Ho MW. GM food animals coming. Science in Society 32,
5. Ho MW. FAQ on genetic engineering. ISIS Tutorial.
6. Definition of Prion, MedicineNet.com,
7. Prion, Wikipedia, http://en.wikipedia.org/wiki/Prion
8. Ho MW. Living test for mad cow disease. Science in Society 28, 5-6, 2005.
9. Weissmann C and Flechsig E. PrP knock-out and PrP transgenic mice in
prion research. Br Med Bull. 2003; 66, 43-60.
10. Criado JR, Sanchez-Alavez M, Conti B, Giacchino JL, Wills DN,
Henriksen SJ, Race R, Manson JC, Chesebro B and Oldstone MB. Mice devoid
of prion protein have cognitive deficits that are rescued by
reconstitution of PrP in neurons. Neurobiol Dis. 2005 Jun-Jul;19(1-2):255-65
11. Denning C, Burl S, Ainslie A, Bracken J, Dinnyes A, Fletcher J, King
T, Ritchie M, Ritchie WA, Rollo M, de Sousa P, Travers A, Wilmut I and
Clark AJ Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene
and the prion protein (PrP) gene in sheep. Nat Biotechnol. 2001, 9(6),
12. Kuroiwa Y, Kasinathan P, Matsushita H, Sathiyaselan J, Sullivan EJ,
Kakitani M, Tomizuka K, Ishida I and Robl JM. Sequential targeting of
the genes encoding immunoglobulin-mu and prion protein in cattle. Nat
Genet. 2004, 36(7), 775-80.
13. Yagi T, Nada S, Watanabe N, Tamemot H, Kohmura N, Ikawa Y and Aizawa
S. A novel negative selection for homologous recombinants using
diphtheria toxin A fragment gene. Analytical biochemistry 1993, 214, 77-86.
14. Klein G, Powers A and Croce C. Association of SV40 with human
tumors. Oncogene 2002, 21(8), 1141-9.
15. ?No BSE-free cow?, New Scientist 25 January 2007,
16. BSE Enquiry Volume 12, Chapter 10. Organic farming,
17. Loonstra A, Vooijs M, Beverloo HB, Allak BA, van Drunen E, Kanaar R,
Berns A and Jonkers,J. Growth inhibition and DNA damage induced by Cre
recombinase in mammalian cells. Proc Natl Acad Sci U S A. 2001 Jul
18. Ho MW. Terminator technology in new guises, ISIS news 3, December,
19. Ho MW. Terminator recombinase does scramble genomes, ISIS News 7/8,
February 2001, http://www.i-sis.org.uk/isisnews/i-sisnews7.php
20. Schmidt, E.E., Taylor, D.S., Prigge, J.R., Barnett, S. and Capecchi,
M.R. (2000). Illegitimate Cre-dependent chromosome rearrangements in
transgenic mouse spermatids. PNAS 97, 13702-13707.
To unsubscribe from SANET-MG:
1- Visit http://lists.sare.org/archives/sanet-mg.html to unsubscribe or;
2- Send a message to <firstname.lastname@example.org> from the address subscribed to the list. Type "unsubscribe sanet-mg" in the body of the message.
Visit the SANET-MG archives at: http://lists.sare.org/archives/sanet-mg.html.
Questions? Visit http://www.sare.org/about/sanetFAQ.htm.
For more information on grants and other resources available through the SARE program, please visit http://www.sare.org.