Download Minimum length of homology in a donor DNA to facilitate

Minimum length of homology in a donor DNA to facilitate
homologous recombination during ZFN mediated gene targeting
Kiho
1Division
1
Lee ,
Jin-hoi
2
Kim ,
Randall S
1
Prather
of Animal Sciences, University of Missouri, Columbia, MO
2Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea
Introduction
Materials & Methods
• Production of small donor DNAs
To identify a minimum length of homology needed to induce HR during ZFNmediated targeting. Donor DNAs with a series of homology lengths were generated
by first annealing two oligonucleotides containing a short homology, then PCR to
extend the homology (Figure 2). ZFN binding sites were modified and an in-frame
stop codon was introduced to the donor DNA; 35 (A), 85 (B), and 125 (C) bp
homology on each arm.
• Gene targeting by electroporation
For gene targeting, 1 million cells were transfected with ZFN constructs with a each
donor DNA; 200ng each. The cells were electroporated with the constructs at 490 V,
1 msec, 3 pulses using a BTX Electro Cell Manipulator (Harvard Apparatus, Holliston,
MA). ZFNs alone served as control. The ZFN used in the study is shown in Figure 3.
• Quantitative real-time PCR
To identify the rate of gene targeting induced by HR, DNA was isolated from the
transfected cells after three days. Then 50ng of DNA was used for the PCR analysis.
Quantitative real-time PCR was then conducted using IQ SYBR Green Supermix (BioRad Laboratories). The PCR was performed on a MyiQ single color real-time thermal
cycler (Bio-Rad). The program used for amplification included an initial temperature
of 94°C for 2 min followed by 40 cycles of 5 sec at 94°C, 30 sec at 60°C, and 30 sec
at 72°C. Real time fluorescence data was collected during the extension time.
Differences in gene targeting were compared by ANOVA and p<0.05 was considered
significant. Three biological replicates with two technical replication were applied.
Figure 3. Designed ZFNs to target pig CMAH. The ZFNs were designed to
recognize 30 bp. The ZFNs are designed to cause DSB on exon 8 of CMAH.
Results
• The highest targeting efficiency was observed when the donor DNA with 125 bp
homology on each arm was used; 10 times higher compared to the donor with 35
bp homology. No recombination event was detected in the negative control as we
expected. When the PCR products were loaded on a gel, we could not detect a
visible PCR product from the 35 bp homology group. Also, highest intensity was
detected from PCR product from the donor with the longest homology (donor C).
A
Relative targeting efficiency through HR
Zinc-finger nucleases (ZFNs) can induce targeted mutations by causing double strand
breaks at a specific location on a chromosome (Urnov et al., 2005). CMP-Neu5Ac
hydroxylase (CMAH) is widely expressed on the endothelial cells of many mammals
except humans (both of these genes are pseudo genes in humans). Therefore, this
epitope is a potential porcine target for human endogenous antibodies. Previously
we produced CMAH knock-out (KO) Minnesota miniature pigs using ZFNs with a
donor DNA containing only 800 bp homology on each arm to induce homologous
recombination (HR) (Figure 1). The length of homology in the donor DNA was much
smaller than conventional targeting vectors. The next question we wanted to address
was to identify a minimum length of homology needed to induce HR during ZFNmediated targeting. Identifying minimum length of homology would help us design
donor DNAs for ZFN-mediated KOs and knock-ins.
0.007
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Control
A
B
C
B
Figure 4. Efficiency of gene targeting through HR in ZFN-mediated targeting of
CMAH. (A) Donor with the longest homology resulted in the highest efficiency of
targeting (donor C). (B) Targeting through HR was detected from donor A using
quantitative real-time PCR, however, we could not detect the PCR product on a gel.
Different letters indicate statistical difference (p<0.05).
Conclusions
• Our results suggest that a minimum of 85 bp on each side of donor DNA is
needed to successfully facilitate HR during ZFN-mediated targeting and longer
homology can be more effective. Further studies may suggest more specific
requirement of homology length during ZFN-mediated targeting.
• Use of donor DNA can introduce a specific mutation rather than KOs caused by
ZFN-mediated NHEJ. This strategy can be used to induce substitution of amino
acids.
Figure 1. Genetically engineered pigs lacking CMAH. Males are monoallelic
KO of CMAH and do not have any integration of exogenous DNA. Females
have biallelic KO of CMAH. Relatively small donor DNA was used for the
production.
References
• Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH,
Gregory PD, Holmes MC. Highly efficient endogenous human gene correction using designed
zinc-finger nucleases. Nature. 2005 435(7042):646-51
• Kwon DN, Lee K, Kang MJ, Choi YJ, Park C, Whyte JJ, Brown AN, Kim JH, Samuel M, Mao J,
Park KW, Murphy CN, Prather RS, Kim JH.Production of biallelic CMP-Neu5Ac hydroxylase
knock-out pigs. Sci Rep. 2013;3:1981. doi: 10.1038/srep01981.
Acknowledgements
Figure 2. Schematic design of ZFN-mediated targeting of CMAH using small
donor DNAs. HR junction was amplified by PCR. Green indicates homology
region. Blue indicates location of modified ZFN binding site with added in
frame termination codon. Three different donors are generated for the
study (A, B, and C).
This work was supported by Woo Jang-Choon project (PJ007849) from the Rural Development
Administration (RDA), Republic of Korea, and Food for the 21st Century at the University of
Missouri.