role of the transgenic animals in drug discovery advantage and limitation process of transgenesis ethics issue covid19 animal model

1. Speaker-Dr Kalpana
16/11/2021
Transgenic Animal Models & their
Applications as screening models

2. outline
Introduction
Historical background
Production of transgenic animals
Application
Ethical issues
Limitation of transgenic animals
References

3. Introduction
 Transgenic animal-- those animals in which genetic arrangements are
changed by insertion of foreign genes
Modified animals are called transgenic animals
DNA that is introduced called transgenic DNA
Process of introducing transgenic DNA called transgenesis

4. For what reason?--TA
Powerful tool for development of disease models
Help develop new drugs and new way for repairing defective genes (“gene
therapy”)
 Provide organs and tissues for use in human transplant surgery
To enhance livestock improvement programs
 Better specificity of models, also lead to a reduction in number of animals used
 Benefit to human health, in economic and efficient production of important
pharmaceutical proteins

5. History
Ralph L. Brinster
[American geneticist ]
(1982)
 First transgenic animal was a ‘super mouse’ by Ralph Brinster and Richard Palmiter in 1982
 Created by human growth hormone gene in mouse genome(1981)
 Model larger than parents
Richard Palmiter

6. Timeline illustrating some animal species used as models

7. Recent animal used for study

9. CREATING TRANSGENIC ANIMALS
a. Microinjection-- Most common techniques
b. Embryonic Stem Cell Transfer
c. Retrovirus-Mediated Gene Transfer

10. DNA Microinjection- Gordon and Ruddle, 1981

11. Benefits of DNA microinjection
 Very simple inexpensive and applied to variety of species and more size of DNA
can be inserted into recipient animal
 Limitations of DNA microinjection
 Success rate low and technique cannot be used into at later
development stage
 Manipulations of oocytes/ embryos/ disruption of parental DNA at
integration also influence normal development of transgenic animal

12. Retro- Virus mediated Gene Transfer-Jaenisch, 1976
Implant to foster
mother

13. Benefits of Retroviral gene transfer method
 Simple,small piece of DNA can be integrated with minimal disruption of host
DNA
 Readily integrates and passes through germ lines allowing for their propagation
into subsequent generations
 Limitations of retroviral gene transfer method
 Unstable to insert large size of foreign DNA
 Risk of retroviral contamination, losing of regulatory sequences, genetic change
other cells without desired addition

14. Embryonic stem cell mediated gene transfer
Gossler et al., 1986

15. Benefits of Embryonic stem cell technique
 Relatively efficient in homologous recombination in comparison to other animal
cells
 Helps to detect precisely mutations in gene via homologous recombination
Limitations of embryonic stem cell technique
 Production, characterization and maintenance of pluripotent embryonic
stem cells lines very difficult

16. Screening of transgenesis animals
Test whether cells incorporate transgene or not, ---
Southern blot assay -- most widely used for testing presence of transgene in host
animals
 Western blot-- detect transgenic protein produced by animals
Enzyme-linked immuno-absorbent assay-- measuring the number proteins in
serum, blood, and urine

17. Application
In medical research TA --used to identify function of specific over or under
expression of gene
Model of human disease
Polio virus
In toxicology biology
In molecular biology –analysis of regulation gene expression and related
changes
In biotechnology -
Cloning procedure
Xenografting
Animal model for drug screening ,
Pharmaceutical industry

18. Some Examples of Transgenic Animals
 Super fish -Growth hormone gene inserted into fertilized egg Increased growth
and size
Glo fish --Genetically modified zebra fish
 Produced by integrating a fluorescent protein
Enviro pig - created by introducing phytase gene of Ecoli
Pig for organ transplant - decrease chance of organ rejection by human body
 Transgenic Sheep
Transgenic Monkey
“ANDI”- transgenic monkey, born in 2000
ROSIE” - first transgenic cow , born in 1997

19. Knockout/Knock in Mice
 First knockout mouse by mario R. Capecchi, martin evans, and oliver smithies in
1989 Nobel prize in physiology or medicin(2007)
A knockout mouse is a genetically engineered mouse in which one or more genes
have been turned off through a gene knockout
 Important animal models for studying role of genes which have been
sequenced, but have unknown functions by causing a specific gene to be inactive
in mouse, and observing any differences from normal behavior or condition,
researchers can infer its probable function

20. KNOCKOUT VS. TRANSGENIC MOUSE?
Knockout: -Gene inactivated /delete specific gene
 to study loss of function
Transgenic: - gene that is integrated into its genome in a random fashion it can
be A foreign gene ,Extra copies of endogenous gene /mutated endogenous gene

21. Transgenic animal model Disease
Zucker fatty rat Hyperinsulinemia obesity
Goto kakizaki rat DM
TGR model (addition od additional
Ren2gene)
Hypertension
Mongolian Gebrills GTCS
NER/Noda epileptic rat Absence seizure
PRKAG2 overexpression WPW syndrome
HPRT mice Lesch-Nyhan disease

22. Transgenic animal model Disease
AKA mice Type 2 diabetes
Insulin receptor substrate-1 (IRS-1)
knockout
Type 2 diabetes
Rat insulin promoter (RIP), Ngn3 and
Pdx-1
Type 2 diabetes
Amyloid-β Transgenic Mouse Models Alzheimer’s disease
Tau Transgenic Mouse Models Alzheimer’s disease
Tg2576(Hamster PrP) Alzheimer’s disease

23. Transgenic animal model Disease
Onco-mouse Cancer
SCID mice AIDS
B2m
beta-2 macroglobulin
AIDS
Drosophila fly Parkinson’s disease

24. Animal model use for covid19
hACE2 TA mice Golden hamster/syrian Ferret Rhesus macaques
Pandey k et al.Transbound emerg Dis 2020
 129 SVEV
 HACE2
 BALB/C
 STA1 gene knockout mice

26. Laboratory for Transgenic animals
• India's first transgenic green mouse has been developed at the Indian
Institute of Science (IISc), Bangalore
Indian laboratory

27. Limitations of Transgenic animal
Insertional mutations resulting in alteration of important biological processes
 Unregulated gene expression resulting in improper expression of gene products
Possibility of side effects in transgenic animals like arthritis, dermatitis and cancer
etc
long term effects on environment when transgenic animals are released into
field
 Abnormalities suffered are more, Reduced fertility, Respiratory circulatory
problems
 Weak immune system
Costly and technically sensitive

28. Ethical Concerns
 Transgene from one species into another species violates “integrity of species”
 Risks to environment, including risk to biodiversity
 Transfer of human genes into animals ( and vice – versa ) dilutes concept of
“humanness”
Use of animals in biotechnology causes great suffering to them
 Biotechnology is disrespectful to living beings , and only exploits them for
benefit of human beings

29. Future of transgenic animals
Current research limited to transferring a small amount of genes at a time
Much work remains to be done for techniques Possible effects of foreign DNA remains a
concern
This will not only increase our understanding of basic biology in
commercial species, but might also lead to the generation of animals that
are more resistant to infectious disease
Decrease time line for drug discovery and laboratory work load

30. Biosafety regulatory –Environment protection act 1986
Recombinant DNA advisory committee
Institutional biosafety committee
Review committee on genetic manipulation
Genetic engineering appraisal committee
State biotechnology coordination committee
District level coordination committee
• Approval from CPCSEA and GEAC IEC
• Lab follow biosafety level 3&4

31. References
Use of laboratory animals in medical research: A brief overview
Animal use in pharmacology education and research: the changing scenario
dinesh K. Badyal, chetna desa
Use of transgenic animals to improve human health and animal production L-M
houdebine
Transgenic animals and their applications shet masih, pooja jain, rasha el baz
Modern approach of transgenic animalsand their applications in prospect of
biotechnology-a review. Available from[accessed oct 26 2021
Animal models in pharmacology: A brief history awarding the nobel prizes for
physiology or medicine catarina V. J (pharmacology 2021;106:356–368)
Transgenic and Infectious Animal Models of HIV-Associated Nephropathy Paul
Rosenstiel

32. References
Vogel H. Pharmacological assays. Drug discovery and evaluation. 2021:1095-1125
The Construction of Transgenic and Gene Knockout/Knockin Mouse Models of
Human Disease Alfred Doyle,1 Michael P. McGarry, 2011 Jul
29. doi: 10.1007/s11248-011-9537-3
Pharmacological Screening Methods & Toxicology: Revised & Updated Kindle
Edition by Avanapu Srinivasa Rao
Postgraduate Pharmacology 2020 by Sarkar Sougata

33. Thankyou

37. Defining Criteria for Animal Model Validation
Predictive validity
 Measure of how well a model can be used to predict currently unknown aspects of disease in humans ( 6-OHDA
rodent model for parkinson's disease
Face validity- how well a model replicates disease phenotype in humans (MPTP non-human primate model for
parkinson's disease)
Construct validity- how well mechanism used to induce disease phenotype in animals reflects currently
understood disease etiology in humans(Smn1 and hsmn2 transgenic mice for spinal muscular atrophy)
CCAC Guidelines and Animal Welfare
The CCAC Guidelines on Transgenic Animals
ACC Protocol Review
Guidelines for Principal Investigators Working with Transgenic Animals The creation, generation, breeding and
propagation of transgenic animals are covered under Section III-D-4 of the NIH Guidelines. These activities are
not exempt from the NIH Guidelines and must be reviewed by the Institutional Biosafety Committee (IBC)

Janell Richardson, PhD
Wednesday, January 2, 2019