Presented by Christian K. Tiambo at the AABNet webinar series, 27 July 2023

1. Better lives through livestock
Stem cell and genetic engineering technologies
for conservation and sustainable development
of African animal genetic resources
Christian K. Tiambo
Scientist - CTLGH/ILRI
Lead - Poultry Cellular Resources, Functional Genomics Tools and Biotechnologies
ABS Nagoya Protocol Officer
c.tiambo@cgiar.org
AABNet webinar series, 27 July 2023

2. 2
Science, 2019

3. 3
Outlines
1. African Population growth and food security needs
2. African Animal Genetic Resources
3. Animal stem cells
4. Genome editing: history and evolution
5. Potential of stem cells and gene editing technologies for
the development of African AnGR

4. 4
1. African Population growth and food security needs
Despite tremendous economic growth in the last decades, Africa is still challenged:
• African population grows and
transforms very fast.
• Africa's total population would
reach nearly 2.5 billion by 2050.
• Need of urgent food assistance
in Africa (million people) FAO,
2020

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1. African Population growth and food security needs
• Expected demand increase by 2050 (world bank, 2020)
Grains: 190%
Meat: 327%
Dairy products: 270%
egg: +++%
• The future of African livestock will influence the
development of the entire continent.
• Urgent need to build the foundations to steer African
livestock industry on a sustainable growth and
development trajectory.
Enahoro et al., 2021

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2. African Animal Genetic resources
1
• Taxonomy and evolution ?
2
• Domestication process and
adaptation?
3
• Management and
conservation plans?
Genes?
Individual
animals ?
Breeds or
Populations
Species ?
There is urgent need for us, to design research to suit our context so that we can get the real picture of (what: genetic asset) we have (Where: on
African continent), and (how: harness its potential locally) to (why: meet the nutritional needs) of (when: present and future) (who: generations).
How much do we know about the African AnGR?
A policy initiative that explores the possible
futures of the African livestock sector,
How can we unlock the genetic potential of
the existing local livestock breed types?
• Great diversity
• Variation of ecosystems.
• Adaptability traits, harsh environment
• Small scale, low inputs
• Extensive or semi-intensive, pastoral
• Low skill, no specialization

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2. African Animal Genetic resources
Stem cells technologies and genetic
engineering are poised to bring a tremendous
transformation of livestock breeding in Africa
for the better in the next few years

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3. Animal stem cells
SCs have acquired a foremost share in all fields of biological research work in veterinary
science, agriculture, human medicine, wildlife conservation and health, etc.
SCs are classified by their source as:
a) Embryonic Stem Cells (ESCs): derived from the inner cell mass (ICM) of blastocyst-stage
embryos
b) Adults stem cells: undifferentiated cells from specific differentiated tissues that can self-renew or
generate new cells to replenish dead or damaged tissue
c) induced Pluripotent Stem Cells (iPSC): pluripotent cells created by reprogramming
differentiated cells.
Stem cells (SCs) are those cells which have two important properties: self-renewal and
differentiation capacity

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3. Animal stem cells
Embryonic stem cell establishment and characterization in different livestock species

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3. Animal stem cells
Ectoderm Mesoderm
Endoderm
Muscles
cells Blood cells
Epithelium
Epithelial
cells
Blastocyst
Zygote
Totipotent
stem cells
Inner cell mass
Pluripotent
stem cells
Fetus
Morula
Embryo
Gastrula
Unipotent
cells
Multipotent
and
oligopotent
cells
Induced Pluripotent
stem cells
Somatic cells
Reprogramming factors
(Oct4, Sox2, Klf4, and c-Myc
(OSKM)
Tissues and
organs with
somatic cells
Differentiation
and
specialization

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3. Animal stem cells
Applications and future usages of induced pluripotent stem cells (iPSCs) from ruminant livestock.
Weeratunga et al., 2023

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4. Genome editing: history and evolution
Since the generation of the first transgenic mouse in 1974 there have been concerted efforts
to genetically modify the genomes of livestock species (Whitelaw and Lillico. 2022)
History of 38 years of GEd livestock
featuring some of the well-known
celebrities in the field.
Alvane, 2020

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4. Genome editing: the tools
Overview of current genome editing tools, ZFN (A), TALEN (B), CRISPR-Cas9 system (C) creating double-strand
breaks (DSB) within the targeted DNA sequence.
Matsumotoab and Nomura, 2023
Clustered
regularly
Interspaced
Palindromic
Repeat CRISPR-
Associated-9
Transcription
Activator-Like
Effector Nuclease
Zinc-Finger
Nuclease
(Non-Homologous End Joining: error-prone)
(Homology-Dependent Repair): faithful
Bjoern Petersen, 2017
single-stranded
oligonucleotide (ssODN)
or double-stranded
Plasmid DNA, Specific
modifications of the
genome
Small insertions or
deletions (Indels) and
subsequent gene
disruption

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4. Genome editing: tools improvement
Matsumotoab and Nomura, 2023
Improvements of CRISPR/Cas9
• Cas9 Nickase (nCas9)
• Dead Cas9 (dCas9)
• Base Editing
• Point Mutation Introduction
• Cas9 Tethering ssODN
• Prime Editor
• Chemically Modified ssODN
• Targeted Integration of Long dsDNA
ZFN TALEN CRISPR-Cas-9
Construction
Protein engineering for
every single target
Protein engineering for
every single target
20-Nucleotide sequence of
single-guide RNA (sgRNA)
Target sequence
recognition
Zinc fingers protein,
protein-DNA interactions
Repeat variable diresidues
(RVDs) repeats, protein-DNA
interactions
sgRNA, RNA-DNA
interactions
Endonuclease
FokI FokI Cas9 and its different
variants
Flexible localization Limited Average Almost total
Endonuclease
construction
3–4 Zinc fingers domains,
slightly laborious
8–31 RVD repeats, slightly
laborious
sgRNA synthesis or cloning,
simple
Delivery
Two ZFNs around the
target sequence
Two TALENs around the
target sequence are
required
sgRNA complementary to the
target sequence with Cas9
protein
DNA sequence
recognition size
(9 or 12 bp) × 2 (8–31 bp) × 2 17–20 bp + NGG × 1
Targeting efficiency Low Moderate High
Affordability / cost
Resource intensive and
time consuming
Affordable but time
consuming
Highly affordable and rapid
In vitro testing slightly laborious slightly laborious Simple
Off-target effects Moderate Moderate High
Multiplexing No No Yes
Time investment High Moderate Low
Janik-Karpińska, et al., 2020

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4. Genome editing: use of CRISPR/Cas9
CRISPR/Cas9 gene editing using either zygote micromanipulation (electroporation or microinjection)
or somatic cell nuclear transfer (SCNT) for generation of livestock animals for various applications.
Perisse et al., 2021

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5. Potential of stem cells and gene editing technologies for the
development of African AnGR
Stem-cell and gene editing breakthrough could preserve diverse African
livestock interests:
• Species and Breeds for conservation,
• Restoration of threatened and extinct breeds
• Development of animal with desire traits
• Accelerated dissemination of elite animal populations
• Animal welfare
• Support to 3Rs ethical research
• Etc.

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5. PGCs and PSCs/iPSCs, Genetic engineering and improvement
Flowchart for trait discovery and trait deployment using NGS approaches for achieving higher genetic gains and accelerated development of improved small ruminants' populations.
Embryonic or
somatic cell
harvesting

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5.1 Stem cells and genetic engineering for conservation, breeds’ restoration, and
dissemination of elite and locally adapted poultry genetics
Biobanking and characterization of tropical indigenous chicken cell lines, Production of chimeras and sterile surrogate host
(DDX4 KO and iCaspase-9 ) for restoration of locally adapted poultry biodiversity and dissemination of potential elite lines
Sterile male and female chicken eggs have been implanted with
reproductive cells from donor chicken, and the resulting chickens
mated together, to produce chicks of the donor breed

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Eg: of conservation and resuscitation of Kenyan locally adapted and improved poultry genetics and chimera
production
Harvesting of chicken PGCs
Chimera hens showing some
phenotypic characteristics of the local
donor at KAIRO Naivasha
Biobanking at ILRI Biorepository
Injection into the recipient embryo and
production of Chimera
Gonads biobanked at ILRI
Counties Male Female Total
Laikipia 44 43 87
Bungoma 9 18 27
Kilifi 4 1 5
Kakamega 14 13 27
Bomet 1 1 2
Homabay 21 19 40
Siaya 13 5 18
Kwale 0 1 1
Baringo 36 40 76
Isiolo 8 7 15
Marsabit 15 12 27
Samburu 18 15 33
KC1 132 96 228
KC2 138 92 230
Total 453 363 816
NBA inspecting the facilities allocated by KALRO to
host the Surrogate host and the ILRI’s backup flock
KALRO leadership visiting the Biobanking Lab
• Local chicken biobanking work in progress
• KALRO will host the CTLGH/ILRI non transgenic surrogate host for
large scale dissemination
• Biobanking and revival collaboration to be extended to other local
AnGR in Kenya using PSC/iPSCs
Counties cell lines biobanked
Nairobi 7
Narok 14
Bomet 42
Siaya 23
Migori 20
Homabay 8
Kakamega 13

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Adapted from Oatley at al., 2017
Advantages
• Indigenous germ line ablated-male (bucks, boar, bull, etc.) carry the sperm of elite male breeds
• Instead of having one elite top male, we would have a thousands
• This provides a transformative step change to disseminate elite semen without changing the existing infrastructure
Kumar et al., 2021 doi: 10.4252/wjsc.v13.i1.1
The Surrogate Buck
The Surrogate boar
Production of Super dads
Eg: of conservation of locally adapted cattle and pig genetic resources, and potential for surrogate sire technology

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E.g of surrogate livestock

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Potential application in wildlife
Credit: Ami Vitale
Sudan: the world’s last
male northern white
rhino, died at 45-year-
old at Ol Pejeta
Conservation of endangered species of wildlife and revive the extinct ones

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Involvement of pluripotent stem cells in
the reproductive cell cycle through
reprogramming, differentiation and
development.
5.2: Reproduction: in vitro gametogenesis (IVG)

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Overview of in vitro gametogenesis (IVG) developments in mice and the current state of IVG in livestock species.
EpiLC: epiblast-like stem cells;
ESC: embryonic stem cells;
PGC-LC: primordial germ cell-like cells.
Botigelli et al., 2023.
5.2: Reproduction: in vitro gametogenesis (IVG)

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5.2: Reproduction: in vitro breeding (IVB)
Strategy, estimated times, and possible alternatives for its
implementation in animal production systems. NT: nuclear transfer.
Goszczynski et al., 2019
Estimated benefits of IVB in comparison with conventional GS
on the cumulative selection response over 25 years
Goszczynski et al., 2019

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5.3: Robust health: Disease resistance
• Disease-resistant genetically modified animals
• Potential animal welfare benefits
Aminul et al., 2020

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Schematic representation of Mzima Cow
5.3: Robust health: Disease resistance
Chicken resistant to bid flu

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5.4: Efficient growth: silencing myostatin gene, etc.
Myostatin (MSTN) negatively regulates
skeletal muscle growth and mutations in
myostatin cause double-muscled phenotype
in various animals.

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5.5: Milk quality and quantity: mineral and organic content
The GnEd offers the opportunity to introduce SNPs associated with milk yield in
the genome of thousands of embryos in just one generation.
Generation and identification of the DNA-free BLG bi-allelic knockout cow.

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5. Adaptability: heat tolerance

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1. Modulating forages and/or rumen
archaea themselves in a manner that
would reduce methanogenesis
2. Selection and breeding for high
productive and low CH4 emitting
animals
3. Vaccines to reduce CH4 production in
the rumen
5. Reduced environmental footprint
Subedi et al., 2022

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5.6 other traits
1. Athletic performance
2. Docility,
3. Environmental fit
4. Ornamental phenotypes
5. Etc.

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5. Genetic entrepreneurship

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Concluding remarks
1. Advances in Animal stem cell and genetic engineering technologies have lot of potentials
for livestock and wildlife development in Africa.
2. The only challenges could be our mindset to bridge the fields of biotechnologies,
capacities, regulations, with sustainable intensification and conservation and keep pace
with innovation.
3. Increased public-private partnership and participatory approach will be essential to
develop innovations and build public acceptance of biotechnologies applications.
4. With the right genetic tools, trained personnel and dedicated collaboration, we may be
able to address many challenges in Africa.

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Attend to core African capacity
… the benefits from science, technology and innovation
have been enjoyed by few, instead of being seen as tools
for the development of all. ….
Today Africa’s leaders view science, technology and
innovation as critical to human development, global
competiveness and ecological management.
– Calestous Juma, 2007
2007
2013
Calestous Juma (1953 – 2017) – had already set the stage …
Mindset: Innovations +++

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Acknowledgement
ILRI - Kenya The Roslin Institute - UK
Christian Tiambo, Scientist, Lead CR-FGT & Biotech., ABS
Nagoya Protocol Officer
Moses Ogugo, Research Officer
Christine Kamidi, Joint Appointee ILRI-KALRO
Triza Tonui, Research Associate – reproductive Technologies and
Biobanking
Sally Katee, Research Associate – ABS Nagoya Protocol, Legal,
Ethics and Biosecurity/Biosafety Compliance
Mike McGrew, Chair of Avian Biotechnologies
Simon Lillico, Research Fellow
Tom Burdon, Group Leader/Senior Research Fellow
Tuanjun Hu, Research fellow
Jon Riddell, research Fellow
Musa Hassan, Chancellor's Fellow
Bruce Whitelaw
Director - The Roslin Institute
Appolinaire Djikeng
Director General - ILRI

37. This presentation is licensed for use under the Creative Commons Attribution 4.0 International Licence.
better lives through livestock
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ILRI thanks all donors and organizations who globally supported its work through their contributions to the CGIAR system