CLONING%20AND%20GENETIC%20MODIFICATION%20OF%20CATTLE%20FOR%20PRODUCTION%20OF%20HUMAN%20ANTIBODIES

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APPLICATION OF TRANSGENIC TECHNOLOGY IN ANIMAL
AGRICULTURE
Hematech, LLC Jim Robl President C.S.O.
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OVERVIEW

• Transgenic technologies
• Pronuclear microinjection
• Transgenic cell cloning
• Applications
• Agriculture
• Human medicine

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NORMAL REPRODUCTIVE PROCESS
EGG
PRONUCLEAR EMBRYO
SPERM
BLASTOCYST Day 7
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PRONUCLEAR MICROINJECTION
Direct microinjection of a few thousand copies
of gene into a pronucleus of a recently
fertilized embryo
LIMITATIONS OF PRONUCLEAR MICROINJECTION

• Gene integration into host DNA is random
• Expression of gene is variable depending on
  integration site
• May insert into an important gene and cause a
  harmful mutation

• Efficiency is very low
• Need to inject 1,000 embryos to produce one
  transgenic calf
• Only a few calves will carry the gene
• Still fewer will pass the gene to offspring

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IN VITRO EMBRYO PRODUCTION
In vitro maturation
EGG
PRONUCLEAR EMBRYO
In vitro fertilization
SPERM
In vitro development
BLASTOCYST
Large numbers of embryos can be produced in
vitro at very low cost
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Eyestone (1999)

• 36,530 pronuclear embryos injected
• 1,472 embryos transferred into recipients
• 226 calves produced (17)
• 18 calves transgenic (8)
• Transgene transmission varied from 3 to 54 among
  bulls

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EMBRYO CLONING
EGG
PRONUCLEAR EMBRYO
SPERM
S
BLASTOCYST
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TRANSGENIC CELL CLONING
PRONUCLEAR MICROINJECTION VS TRANSGENIC CLONING

• Gene integration into host DNA is random
• Expression of gene is variable depending on
  integration site
• May insert into an important gene and cause a
  harmful mutation

• Efficiency is very low
• Need to inject 1,000 embryos to produce one
  transgenic calf
• Only a few calves will carry the gene
• Still fewer will pass the gene to offspring

• Efficiency is moderate
• Need to manipulate 100 embryos to produce one
  transgenic calf
• All calves will carry the gene
• Most will pass the gene to offspring

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TRANSGENIC CLONING TECHNOLOGIES

• Gene Targeting DNA sequence is inserted into a
  specified site in the host DNA
• Knock out the function of genes or replace genes
• Insert genes into sites that do not cause harmful
  mutations
• Predictable expression
• Microchromosome Transfer Insertion of a very
  long sequence of DNA
• Microchromosome can carry large complex genes or
  many genes

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CONCLUSION

• Transgenic technologies
• Pronuclear microinjection
• Transgenic cell cloning
• Applications
• Agriculture
• Human medicine

Transgenic technology is no longer a significant
impediment for producing transgenic beef cattle
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TRANSGENIC APPLICATIONS IN AGRICULTURE

• Dairy Cattle Brophy et al. (2003)
• Inserted extra copies of beta or kappa casein
• 8 to 20 increase in beta casein
• Two-fold increase in kappa casein
• Swine Pursel et al. (1999)
• Inserted an insulin-like growth factor gene
• Reduction in backfat and increase lean in gilts
• Swine Nottle et al. (1999)
• Inserted a growth hormone gene
• Increased live weight gain and feed efficiency
  and reduced backfat thickness

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FACTORS IMPACTING THE DEVELOPMENT OF TRANSGENIC
BEEF CATTLE

• Regulatory issues
• FDA
• Food Safety
• Environmental impact
• Animal welfare
• Business model
• Lack of integration in the beef industry
• Producer and consumer acceptance
• How do you generate a profit
• Genetic factors
• Zygosity Are homozygous males and females needed
  to pass on trait
• Insert site Need defined insert site

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CONCLUSION

• Applications for transgenic cattle
• Agriculture
• Human medicine

Great long term potential but currently
significant impediments of market entry
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PRODUCTION OF HUMAN THERAPEUTIC DRUGS IN BOVINE
MILK

• Eyestone et al.,1999
• Human alpha lactalbumin
• Behoodi et al., 2001
• Human serum albumin
• Van Berkel et al., 2002
• Human lactoferrin
• Chen et al., 2002
• Human bile salt stimulated lipase

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THE HEMATECH HUMAN ANTIBODY PRODUCTION SYSTEM
Tc COW Designed to produce human antibodies in
response to immunization
Anti- Virus
Virus
Anti- Bacteria
Collect Plasma
Immunize Tc Cow
Bacteria
Anti- Toxin
Toxin
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WHAT ARE ANTIBODIES?
Blood Donation
Blood Serum Containing Antibodies
Serum
Y
Y
Y
Y
Y
Y
Y
Y
Y
Clot
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y

• Antibodies can be used to treat
• Infections
• Cancer
• Organ transplant rejection
• Autoimmune disease

Y
Y
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STEPS FOR MAKING A HUMAN ANTIBODY-PRODUCING COW

• STEP 1
• Introduction of human antibody genes
• Very large genes
• Two genes needed
• Microchromosome technology
• STEP 2
• Inactivation of bovine antibody genes
• Multiple genes
• Two alleles must be inactivated for each gene
• Gene targeting technology

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INTRODUCTION OF HUMAN ANTIBODY GENES USING A
MICROCHROMOSOME VECTOR
Microchromosomes are independently replicating
vectors with very large DNA capacity (millions
of DNA bases)
Chr 14
Chr 22
Heavy chain gene
Chr 14 fragment
Chr 22 fragment
Lambda chain gene
Kuriowa et al. 1999 (Nature Biotech)
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CALVES CARRYING HUMAN ANTIBODY GENES IN A
MICROCHROMOSOME
Calves with human antibody genes produce low
levels of human antibody
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CALVES WITH BOVINE ANTIBODY GENES KNOCKED OUT
One allele of one gene knocked out Heterozygous
knockout
Two alleles of one gene knocked out Homozygous
knockout
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ANIMAL PRODUCTION SYSTEM
Source Herd
Recipients need to be obtained from known
sources, preferably closed herds produced
under strict disease control and
monitoring standards
Quarantine Center For Recipients
Source Herd
Disease break
Source Herd
Embryo Transfer Center with Recipient Pool
Disease break
Disease break
Pregnant Recipients Maintained to 245
days Gestation
Calving Center
Production Center
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CONCLUSION

• Applications for transgenic cattle
• Agriculture
• Human medicine

Potential for salaried positions in the cattle
industry and for production of qualified
recipients and feed