This document discusses the future of innovations in transgenic animals through genome editing and reproductive technologies. It outlines various techniques for creating transgenic animals such as embryo splitting, pronuclear injection, somatic cell nuclear transfer (SCNT), and molecular tools like recombinases, transposons, ZFNs, TALENs, and CRISPR/Cas9. Applications of transgenic animals include disease modeling, producing therapeutic agents, improving traits like growth and milk production, generating disease resistance, and xenotransplantation. While progress has been made, challenges remain around efficiency, mosaic mutations, and off-target effects that further research and regulation may help address.

1. FUTURE OF INNOVATIONS IN TRANSGENIC ANIMALS
PRESENTED BY:
DR. ALISHA
L-2021-ABT-01-D
DR. KANCHAN

2. TRANSGENIC ANIMALS
• GENOME EDITING- MODEL ORGANISMS
• BIOTECHNOLOGICAL IMPROVEMENTS-
a) WELL BEING
b) NOURISHMENT
c) REARING
d) REPRODUCTION

3. IMPORTANCE
APPLICATIONS:
1. Disease pathogenesis
2. Novel therapeutic agents
3. Novel treatment regimens
4. Xenotransplantation
5. Bioreactors
6. Production of disease resistant animals
7. Genetically superior animals
8. Investigation of gene functions Kalds et al., 2019

4. PATHOGENESIS-
• Animals as models for
human diseases
• Pigs-Human atherosclerosis; LDLR. Double-
knockout pigs
• Non-human primates- Lentivirus
based Huntington disease- Brain and
behavioural defects
Aggarwal, et. al., 2021
Zhao et. al., 2019

5. XENOTRANSPLANTATION
• ORGAN TRANSPLANTATION-End stage organ
failure
• Shortage of organ donors
• Several organ/cells/tissue donor models
a. Pig
b. Chimpanzee
• Limitations:
a. Immune rejection
b. Potential cross-species infection
Zhao et. al., 2019

6. BIOREACTORS
• Bacteria and Yeast: Limited applications
• Transgenic animals as bioreactors
• Produce proteins-Economic and efficient
manner
• Mammary gland as a high protein
producing factory, beta-globulin

7. DISEASE RESISTANT ANIMALS
• SiRNA based suppression of PrPc gene
modification for resistant animals
against Prion disease
• Transgenic mice-human enteric alpha-
defensin peptide-small intestine crypts-
Salmonella typhimurium
Lassnig and Muller, 2015

8. TECHNIQUES
• Reproductive biotechnology
• Molecular techniques
Kalds et al., 2019

9. REPRODUCTIVE TECHNIQUES
2015, Encyclopedia Brittanica

10. THE FIRST WAVES
• Embryo splitting-
a. Cloning by embryo splitting-Twinning by
separation of blastomeres
b. Willadsen, in 1979.
c. Artificial microsurgical twinning at cleavage or
blastocyst stage
• Limitations:
a) Technical difficulties
b) Sub-optimal pregnancy rates-Limited number of
individuals
c) Limited divisibility-2-4 genetically identical animals

11. PRO NUCLEAR INJECTION
• Introduction of DNA construct -pronuclei of
fertilized eggs
• In 1985, by Hammer.
• Limitations:
a) Low efficiency
b) Random integration
c) Variable copy of integrated constructs
d) Visualization of pro-nuclei

12. THE SECOND WAVE-EMBRYONIC CELL CLONING
• A type of nuclear transplantation
technique
• In 1986, Willadsen
• Use of 8-16 called ovine embryo
blastomere nuclei for transplant with
ovine enucleated metaphase II oocyte
cytoplasts to produce live lambs.
biosci.gatechu.edu

13. SOMATIC CELL CLONING (SCNT)
• Dolly, 1997, Wilmut et al.
• Donor cell nuclei via transfer ion of donor
cell nuclei with DNA expression constructs or
vectors
• Various nuclei donors-
a. Adult mammary gland cells
b. Adult granulosa cells
c. Adult cumulus cells
d. Fetal fibroblasts cells
• Limitations:
a) Low efficiency
b) Potential for developmental anomalies
2015, Encyclopedia Brittanica

14. HANDMADE CLONING
• Simplified version of SCNT
• Procedure
i. Handmade bisection of zona-free oocytes
ii. Staining
iii. Selection of cytoplasts
iv. Fusion of somatic cells with two cytoplasts
v. Equally sized reconstructed embryos
• Advantages
i. Less expertise
ii. Skill
iii. Time
Sylvia Pagan Westphal, 2002

15. MOLECULAR TOOLS

16. RECOMBINASES
• Recombinases-
 Site specific genetic recombination
 Derived from nature
 Interaction between recombinases and their
recognition sites
 Ability to perform deletions, insertions and
inversions in DNA sequences
• Site specific recombinases-
 Cre/loxP
 Flp/FRT
 PhiC31/attP
 Tyr
 Ser

17. TRANSPOSONS AND RNAI
• Transposons
a. Sleeping beauty
b. Piggy Bac
• RNAi
a. Gene Silencing
b. Gene knockout
Mariuswalter, 2017

18. ZFNS, TALENS AND CRISPR
• ZFNs site specific endonucleases- Zinc-finger
proteins and FokI DNA restriction enzymes
• ZFNs target is predetermined sequence, where
site specific modification happen via induction of
DSB repair pathways
• Has been used successfully in sheep and goat, to
produce high meat producing animals by
modifying MSTN gene.
• Limitations:
a. Difficult to design
b. Potential insertional mutagenesis,
c. toxicity, and
d. off-target events
Kalds et al., 2019
Hillary and Caesar, 2021

19. TALENS
Kalds et al., 2019
• TALEs naturally occurring proteins, secreted by plant
pathogenic bacteria Xanthomonas species
• Series of TANDEM repeats,
• dimers,
• consisting of 30-35 amino acids
• Fok1 DNA restriction enzyme
• Can recognize and bind a single nucleotide
• TALEN-mRNA cytoplasmic injection was MSTN-edited
(Proudfoot et al., 2015)
• Fibroblasts with modified MSNT were used as nuclear
donor for SCNT
• Limitations:
a. Difficult to design
b. Potential insertional mutagenesis,
c. toxicity, and
d. off-target events

20. CRISPR
• CRISPR/Cas9 system - derived from prokaryotes which use as
a defence mechanism, first used in mammalian genome,
2013.
• An RNA-directed Cas9 protein and ∼20-nucleotide
sgRNA, which leads the Cas9 protein to a user-defined
DNA target site as long as it is next to a protospacer
adjacent motif (PAM) sequence
• Limitations:
a. Potential insertional mutagenesis, and,
b. off-target events
Ball., 2016

21. Natl Sci Rev, Volume 6, Issue 3, May 2019, Pages 402–420, https://doi.org/10.1093/nsr/nwz013
The content of this slide may be subject to copyright: please see the slide notes for details.
MAJOR STRATEGIES TO RECRUIT DNA- AND RNA-TARGETING AND
MODIFYING ENZYMES VIA THE CRISPR/CAS SYSTEMS
CRISPR/Cas9 system can be applied for
a. Promotion of muscle growth and development
b. Promotion of fibre length and growth
c. Molecular manipulation of milk components
d. Promotion of reproductive performance
e. Generation of disease resistant and disease model
animals
f. Xenotransplantation

22. NEW PROPOSALS/FUTURE DIRECTIONS
• Strategy to generate large founder animals
with a desired allele in one step, without a
prolonged period of breeding, is in high
demand.
• Mosaic mutations, which are commonly
observed in zygote injection-based genome
editing, are another potential challenge in
the editing of large animals.

23. FURTHER EXPECTATIONS
• New genes and genome areas need to be explored using modern DNA editing technology and
reproductive biotechnology tools.
• A controlled and regulated release of fundings need to be in place for continuous growth of this
scientific area.
• With further studies to solve the ‘off-target’ effects and potential risks to the host genome,
genome editing of animals may become more accepted by the public.
• FDA has determined that animals with intentionally altered genomes should be subjected to
regulations under the provisions of new animal drugs.
• Unlike the FDA, the US Department of Agriculture (USDA) has stated that the USDA will not
regulate genetically modified plants produced by the new genome editing techniques, which will
definitely accelerate the commercialization of genome-edited organisms
• Further optimization of the existing genome editing system and the generation of new tools for
precise gene modification will additionally accelerate the development of genetically modified
animals, organs and tissues for agriculture, regenerative medicine and therapeutic applications.