Supplementary Material for
Generation of mastitis resistance in cows by targeting human
lysozyme gene to beta-casein locus using zinc-finger nucleases
Xu Liua,b, Yongsheng Wanga,b, Yuchen Tiana,b, Yuan Yua,b, Mingqing Gaoa,b,
Guangdong Hua,b, Feng Sua,b, Shaohui Pana,b, Yan Luoa,b, Zekun Guoa,b, Fusheng
Quana,b, Yong Zhanga,b,1
This Word file includes:
Figs. S1 to S5
Table S1
Figure S1 Vector maps of gene-targeting vector pCSN2-hLYZ-Neo-EGFP.
Figure S2 Surveyor nuclease assay of the 16 gene-targeted cell colonies at the eight
most-likely off-target sites. Lane 1, DNA marker. Lane 2, non-transfected cells were
used for negative control. Lanes 3–18, 16 gene-targeted cell colonies suitable for NT.
Figure S3 Western blot analysis of human lysozyme production by transfected bovine
mammary epithelial cells. (A) Vector map of construct used for expressing cytosolically
directed human lysozyme and EGFP fusion protein. (B) Vector map of construct used
for expressing secreted human lysozyme and EGFP fusion protein. (C) Vector map of
construct used for targeting CSN2 locus in bovine mammary epithelial cells with ZFNs.
(D) human lysozyme and EGFP fusion protein was expressed in the cells. The bovine
mammary epithelial cells were transfected by pEGFP-C-hLYZ. (E) The expressed
human lysozyme and EGFP fusion protein was secreted into the extracellular. The
bovine mammary epithelial cells were transfected by pEGFP-S-hLYZ. (F) Human
lysozyme and EGFP fusion protein expression was observed 2 days after treatment with
inductive medium. The bovine mammary epithelial cells were transfected by pEGFPI-hLYZ and pZFN1 plus pZFN2. D’, E’ and F’ are the bright field view of D, E and F,
respectively. Proteins in cell extracts (D) or medium (E, F) were probed with antilysozyme antibody, anti-GFP antibody and anti-casein antibody. (G, H, I) Western blot
analysis for the expression of GFP, hLYZ and casein in transfected bovine mammary
epithelial cells. Lane 1, non-transfected cells were used for negative control. Lanes 2–
4, Cells were transfected with plasmids containing Cyto-hLYZ-EGFP (lane 2), Sec-LysEGFP (lane 3), or Ind-Lys-EGFP (lane 4). Lane 5, standard substances of GFP, Lys and
casein were used for positive control.
Figure S4 Total proteins from transgenic cows hLYZ1-5 and non-transgenic cows (WT)
were separated on a 15% polyacrylamide gel and visualized by Coomassie blue staining.
Figure S5 Response of transgenic (n = 5) and non-transgenic (n = 5) cows to
intramammary infusion. (A) Lipopolysaccharide (LPS)-binding protein concentration
in serum. (B) Serum amyloid A circulating concentrations.
Table S1: Theoretical off-target hotspots based on sequence identities to the CSN2
ZFN binding site
Identity to target site
bp ZFN monomer %
16
16
15
15
16
16
15
15
Left-F
Left-F
Left-F
Left-R
Right-F
Right-F
Right-F
Right-R
88.9%
88.9%
83.3%
83.3%
100%
100%
93.8%
93.8%
RefSeq
In/Ex
Chr
Coordinate
ZFN Binding Site
DOCK11
In
In
In
In
In
In
In
In
X
24
24
6
14
14
27
18
1054469
12265288
5760409
3094503
2556274
3221824
4415251
6299052
CATCCACTATCTCAGT
TCATCCACgATCTCAGT
TTCATCCACTtTCTCA
TTCAaCCACTATCTCA
CCTATGGGACCACAAG
CCTATGGGACCACAAG
CTATGGGACCACAAG
CCTATGGGACCACAA
SKOR2
PTPRM
PDGFRA
XKR9
LACTB2
ZNF385D
N4BP1