Presentation on the research paper titled: TALEN-induced homologous recombination in goats directs production of B-lactoglobulin-free, high-human lactoferrin milk.

1. TALEN-induced homologous recombination in goats
directs production of β-lactoglobulin-free,
high-human lactoferrin milk
Chenchen Cui, Yujie Song, Jun Liu, Hengtao Ge, Qian Li, Hui Huang, Linyong Hu, Hongmei Zhu, Yaping Jin & Yong Zhang
Presented by: Group 3
Soon Chee Haw - 0333249
Wan Wai Yan - 0337820
Bryan Lim Wei Xiang - 0331563
Thivaraj Krishnan - 0338079
Palinda Tennekoon - 0337435

2. TALEN-induced homologous recombination in goats directs production of
β-lactoglobulin-free, high-human lactoferrin milk
- Introduction - What they’re doing, why they wanna do it
- Method - How they approach the tools
- Outcome - results+discussion
- Challenges , advantages & disadvantages - discussion
- Current development
- Summary

3. Case Study
● β-Lactoglobulin (BLG) is a major allergen present in goat’s milk
● Genetic modifications using endonucleases are carried out to reduce BLG
levels in goat’s milk
● TALEN-mediated gene knockout followed by gene knock-in is used to create
BLG knockout goats for production of human lactoferrin (hLF)
Introduction

4. Introduction
● TALEN is the acronym of transcription activator-like effector nucleases
● It is a form of engineered endonucleases that allows targeted genetic
modification in livestock
What is TALEN

5. Introduction
● TALEN consists of TAL effector DNA-binding domain linked with FokI
endonuclease
● Each TALE domain is restricted only to one nucleotide and does not affect the
binding specificity of neighboring TALEs
● This confers an advantage by virtue of making TALENs design easier
Why TALEN

6. TALEN mechanism

7. Overview - flow of experiment
● Investigated BLG knockout
● Investigated hLF knockin
● Produced target cells for SCNT
● Mono-allelic goats
● Bi-allelic goats
Knockout of BLG followed by knockin of hLF

8. Methods
● Disruption of BLG gene via TALEN-induced HR
○ TALEN 1/2
● pBLG-neo-M
○ Neomycin resistance gene (Positive selection)
○ Herpes simplex virus type 1 thymidine kinase (Negative selection)
● pBLG-neo
○ 3’ arm replaced with 5.3kb homologous sequence
● Phosphoglycerol kinase promoter (PGK)
● PGK cassette inserted
○ Electroporation
Disruption of β-lactoglobulin (BLG) gene via homologous recombination

9. Methods
Targeting vectors: pBLG-neo M & pBLG neo

10. Methods
● PCR analysis
● pBLG-neo
○ 5’ junction analysis with PCR primers P1/P2
● pBLG-neo-M
○ 5’ junction PCR analysis
○ 3’ junction PCR analysis
● Clones that passed two rounds of PCR analysis subjected to southern blot
analysis
Analysis

11. Methods
● Introduction of hLF gene into BLG locus
● pBLG-hLF-neo
○ hLF cDNA
○ Bovine growth hormone polyadenylation signals
○ Between 5’ arm and selection marker
● Co-electroporation with TALEN1/2
Introduction of human lactoferrin (hLF) gene

12. Methods
Knockin of human lactoferrin (hLF) gene at BLG locus

13. Methods
● Generating target cells as donor nuclei for SCNT
● TALEN-encoding mRNAs
○ Prevents genomic integration of TALEN-DNA constructs
● PCR analysis
● Clones with high blastocyst rates selected
● Cloned goats
○ BLG+/-
○ BLG+/hLF
Generating mono-allelic targeted goats

14. Methods
● BLG+/- fibroblasts isolated from monoallelic goats
○ To target second allele
● pBLG-hLF-puro (secondary vector)
○ Puromycin selection marker
○ Replacement of BLG allele
● TALEN-encoding mRNAs and secondary vectors
○ Used to prepare biallelic cells for SCNT
Producing biallelic cells

15. Methods
Producing biallelic cells

16. Outcome
● NHEJ-mediated gene disruption in goat fetal fibroblasts.
● Cells tranfected with TALENs yielded a good level of fluorescence, proving target
site were cleaved.
● HR-mediated gene targeting by using TALENs in GFFs.

17. Outcome
A) shows targeting strategy with the help of TALENs
B)shows PCR analysis, screening for the targeting event with plasmid pBLG-neo-M.
C)southern blot analysis of junction PCR-positive clones
D) shows a unique 6.4kb band that has PGK-neo cassette targeted integration.
E)sequence comparison of wild type and targeted gene.

18. Outcome
A)Schematic of knock-in-strategy for the BLG Locus
B)Junction PCR analysis for screening the targeting event by using donor pBLG-hLF-neo.
C)southern blot analysis of BLG+/- goats.
D)sequence comparison

19. Outcome (second allele targeting)
A) PCR analysis of Bi-allelic targeted Goats
B) Southern blot analysis
C) Gfp expression
D) Sequence comparison similar to previous outcome

20. A)Milk analysis of mono allelic BLG-targeted goat
B)analysis of BLG through qPCR
C)Analysis of milk from Bi-allelic BLG targeted goat
D) Reverse transcriptase PCR analysis of BLG mRNA from Bi allelic
Outcome (BLG knockout milk analysis)

21. Outcome (discussion)
● Germline transmission of transgenic mutation is vital to ensure a continued
line of transgenic goats
● TALEN-induced modification of zygotic genomes are able to be transmitted
through germline to the next generation through mating BLG+/- goats with
either wild-type or BLG +/- goats
● Gene targeting through TALEN-induced HR has been demonstrated to be an
efficient approach to introduce precise genetic modification
● Gene knockout followed by gene knock-in has generated BLG-free goats that
are able to produce hLF with a stable germline for subsequent generations

22. Challenges
● The use of NHEJ-induced modification gave unpredictable results, and screening
for functional loss mutants was difficult. This was overcome by using HR
modification which was precise.
● Concern of release of antibiotic resistance gene into the environment due to the
integration of selectable marker genes, however to solve this, loxP sites were used
to remove the marker genes.
● Initially pBLG-neo was delivered into GFFs via electroporation but none of the
clones were targeted. In order to solve this TALENs were introduced into GFFs with
targeting vectors.

23. Advantages Disadvantages
● Efficient method of gene
modification
● Reliability of PCR based analysis is
high.
● The modifications are germ-line
transferable
● Multiple site specific modifications
were achieved in shorter time
● Precise gene alterations are not
limited to DBS, can be used for
more extensive applications
● High engineering feasibility.
● Designing the experiment may be a
bit complex due to multiple identical
repeats.
● Requires individual gene tailoring
● Costly
● Lower efficiency compared to
CRISPR.

24. Current development
Exploring the possibilities of goats mammary glands bioreactors – in an industrial scale.
● Bioreactors based on gene-edited ruminants were developed for biomedical materials, such as
enzymes and hormones
● Exogenous genes are routinely fused to the regulatory elements of mammary gland-specific genes
before they are randomly transferred into the animals.
● The expression levels of the same foreign protein usually vary among transgenic animals that have
been generated by the same method.
● In the present study, the mammary glands of precisely genetically modified goats secreted hLF at a
high and stable level.
● The BLG gene replacement with hLF cDNA induced large-scale hLF expression and almost 45%
reduction of BLG expression in the milk from BLG+/hLF goats.
● It is believed that the expression of the intact BLG allele was upregulated to compensate for the
inactive allele in mono-allelic targeted goats; the increased hLF expression also compensated to
balance the milk protein synthesis.

25. Germline transmission of transgenic mutations
● TALEN-induced direct modification of zygotic genomes can be transmitted through the germline to the
next generation
● The modifications were stably transmitted to the progeny by mating BLG+/− goats with the wild-type or
BLG+/− goats -thus paving way for the possibilities of the production of goat mammary gland
bioreactor.
Picture courtesy: Freitas VJF, Melo LM, Batista RITP, Souza-Fabjan JMG, Teixeira DIA, et al. (2014)
Goats (Capra hircus) as Bioreactors for Production of Recombinant Proteins Interesting to
Pharmaceutical Industry. Clon Transgen 3: 130. doi:10.4172/2168-9849.1000130

26. Summary
● In summary, results demonstrated that NHEJ-induced mutations are inefficient in primary cells that resist
dilution cloning.
● Gene targeting through TALEN-induced HR is an efficient approach to introduce precise genetic
modification, which knocks out protein production in the mammary glands of livestock.
● Compared with breeding to produce homozygous animals, sequential gene targeting can provide multiple
genetic modifications for different purposes within a reduced time period.
● Application gene knockout followed by gene knock-in to generate BLG-free goats as mammary gland
bioreactors for the large-scale production of hLF.
● This germline-transmittable genetic modification in livestock is predicted to be a stepping stone in
providing a powerful platform for agricultural or biomedical applications.

27. Reference
Cui, C., Song, Y., Liu, J. et al. Gene targeting by TALEN-induced homologous recombination in goats directs production of
β-lactoglobulin-free, high-human lactoferrin milk. Sci Rep 5, 10482 (2015). https://doi.org/10.1038/srep10482
Khan, S. H. (2019, April 3). Genome-Editing Technologies: Concept, Pros, and Cons of Various Genome-Editing Techniques and
Bioethical Concerns for Clinical Application. Retrieved from https://www.sciencedirect.com/science/article/pii/S2162253119300587
Yeadon, J. (n.d.). Pros and cons of ZNFs, TALENs, and CRISPR/Cas. Retrieved from
https://www.jax.org/news-and-insights/jax-blog/2014/march/pros-and-cons-of-znfs-talens-and-crispr-cas
Yang, P., Wang, J., Gong, G., Sun, X., Zhang, R., Du, Z., … Li, N. (2008). Cattle mammary bioreactor generated by a novel procedure
of transgenic cloning for large-scale production of functional human lactoferrin. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2565487/
Et al, G., 2020. Improving Milk For Human Consumption Through Genetic Engineering Technologies. [online] Core.ac.uk. Available at:
<https://core.ac.uk/download/pdf/162674693.pdf> [Accessed 5 June 2020].