Supplementary Information for “Targeted integration in rat ...Nature Biotechnology: doi: 10.1038/nbt.1731. 16 Supplementary Figure 6 1 25 2 26 3 27 4 28 5 29 6 30 7 31 8 32 9 3310

1

Supplementary Information for “Targeted integration in rat and mouse embryos

with zinc-finger nucleases”

Methods

Full ZFN amino acid sequences DNA recognition helixes are in red. Mdr1a left-hand protein: Mdr1a right-hand protein: PXR left-hand protein:

1 MDYKDHDGDY KDHDIDYKDD DDKMAPKKKR KVGIHGVPAA MAERPFQCRI

51 CMRNFSDNAA LTEHIRTHTG EKPFACDICG RKFA TSSNLS RHTKIHTGSQ

101 KPFQCRICMR KFAQSGDLTR HTKIHTGEKP FQCRICMRNF S RSDTLSQHI

151 RTHTGEKPFA CDICGRKFAD NANRTKHTKI HLRGSQLVKS ELEEKKSELR

201 HKLKYVPHEY IELIEIARNS TQDRILEMKV MEFFMKVYGY RGKHLGGSRK

251 PDGAIYTVGS PIDYGVIVDT KAYSGGYNLP IGQADEMERY VEENQTRNKH

301 LNPNEWWKVY PSSVTEFKFL FVSGHFKGNY KAQLTRLNHI TNCNGAVLSV

351 EELLIGGEMI KAGTLTLEEV RRKFNNGEIN F*

1 MRSDYKDHDG DYKDHDIDYK DDDDKMAPKK KRKVGIHGVP AAMAERPFQC

51 RICMRKFATS GHLSRHTKIH TGEKPFQCRI CMRNFS QSSD LSRHIRTHTG

101 EKPFACDICG RKFAQSADRT KHTKIHTGSQ KPFQCRICMR NFS RSDVLSE

151 HIRTHTGEKP FACDICGRKF A QSGHLSRHT KIHLRGSQLV KSELEEKKSE

201 LRHKLKYVPH EYIELIEIAR NSTQDRILEM KVMEFFMKVY GYRGKHLGGS

251 RKPDGAIYTV GSPIDYGVIV DTKAYSGGYN LPIGQADEMQ RYVKENQTRN

301 KHINPNEWWK VYPSSVTEFK FLFVSGHFKG NYKAQLTRLN HKTNCNGAVL

351 SVEELLIGGE MIKAGTLTLE EVRRKFNNGE INF**

1 MDYKDHDGDY KDHDIDYKDD DDKMAPKKKR KVGIHGVPAA MAERPFQCRI

51 CMRNFSDRSH LSRHIRTHTG EKPFACDICG RKFA TSGNLT RHTKIHTGSQ

101 KPFQCRICMR NFS QSSDLSR HIRTHTGEKP FACDICGRKF A RSDHLTQHT

151 KIHLRGSQLV KSELEEKKSE LRHKLKYVPH EYIELIEIAR NSTQDRILEM

201 KVMEFFMKVY GYRGKHLGGS RKPDGAIYTV GSPIDYGVIV DTKAYSGGYN

251 LPIGQADEME RYVEENQTRN KHLNPNEWWK VYPSSVTEFK FLFVSGHFKG

301 NYKAQLTRLN HITNCNGAVL SVEELLIGGE MIKAGTLTLE EVRRKFNNGE

351 INF*

Nature Biotechnology: doi: 10.1038/nbt.1731

2

PXR right-hand protein: Full ZFN coding sequences

Mdr1a left-hand protein ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATCCACGGGGTACCCGCCGCTATGGCTGAGAGGCCCTTCCAGTGTCGAATCTGCATGCGTAACTTCAGTGACCGCTCCCACCTGTCCCGCCACATCCGCACCCACACCGGCGAGAAGCCTTTTGCCTGTGACATTTGTGGGAGGAAATTTGCCACCTCCGGCAACCTGACCCGCCATACCAAGATACACACGGGATCTCAGAAGCCCTTCCAGTGTCGAATCTGCATGCGTAACTTCAGTCAGTCCTCCGACCTGTCCCGCCACATCCGCACCCACACCGGCGAGAAGCCTTTTGCCTGTGACATTTGTGGGAGGAAATTTGCCCGCTCCGACCACCTGACCCAGCATACCAAGATACACCTGCGGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGGAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACCTCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCTGATAA Mdr1a right-hand protein ATGAGATCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCATGGGGTACCCGCCGCTATGGCTGAGAGGCCCTTCCAGTGTCGAATCTGCATGCGTAAGTTTGCCACCTCCGGCCACCTGTCCCGCCATACCAAGATACACACGGGCGAGAAGCCCTTCCAGTGTCGAATCTGCATGCGTAACTTCAGTCAGTCCTCCGACCTGTCCCGCCACATCCGCACCCACACCGGCGAGAAGCCTTTTGCCTGTGACATTTGTGGGAGGAAATTTGCCCAGTCCGCCGACCGCACCAAGCATACCAAGATACACACGGGATCTCAGAAGCCCTTCCAGTGTCGAATCTGCATGCGTAACTTCAGTCGCTCCGACGTGCTGTCCGAGCACATCCGCACCCACACCGGCGAGAAGCCTTTTGCCTGTGACATTTGTGGGAGGAAATTTGCCCAGTCCGGCCACCTGTCCCGCCATACCAAGATACACCTGCGGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTG

1 MRSDYKDHDG DYKDHDIDYK DDDDKMAPKK KRKVGIHGVP AAMAERPFQC

51 RICMRNFSQS SDLSRHIRTH TGEKPFACDI CGRKFA RSDA LTQHTKIHTH

101 PRAPIPKPFQ CRICMRKFA D RSDLSRHTKI HTGEKPFQCR ICMRNFS RSD

151 NLSVHIRTHT GEKPFACDIC GRKFA DRSNL TRHTKIHLRG SQLVKSELEE

201 KKSELRHKLK YVPHEYIELI EIARNSTQDR ILEMKVMEFF MKVYGYRGKH

251 LGGSRKPDGA IYTVGSPIDY GVIVDTKAYS GGYNLPIGQA DEMQRYVKEN

301 QTRNKHINPN EWWKVYPSSV TEFKFLFVSG HFKGNYKAQL TRLNHKTNCN

351 GAVLSVEELL IGGEMIKAGT LTLEEVRRKF NNGEINF**


3

GGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGAAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACAAAACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCTGATAA PXR left-hand protein ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATCCACGGGGTACCCGCCGCTATGGCTGAGAGGCCCTTCCAGTGTCGAATCTGCATGCGTAACTTCAGTGACAACGCCGCCCTGACCGAGCACATCCGCACCCACACCGGCGAGAAGCCTTTTGCCTGTGACATTTGTGGGAGGAAATTTGCCACCTCCTCCAACCTGTCCCGCCATACCAAGATACACACGGGCAGCCAAAAGCCCTTCCAGTGTCGAATCTGCATGCGTAAGTTTGCCCAGTCCGGCGACCTGACCCGCCATACCAAGATACACACGGGCGAGAAGCCCTTCCAGTGTCGAATCTGCATGCGTAACTTCAGTCGCTCCGACACCCTGTCCCAGCACATCCGCACCCACACCGGCGAGAAGCCTTTTGCCTGTGACATTTGTGGGAGGAAATTTGCCGACAACGCCAACCGCACCAAGCATACCAAGATACACCTGCGGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGGAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACCTCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCTGATAA PXR right-hand protein ATGAGATCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCATGGGGTACCCGCCGCTATGGCTGAGAGGCCCTTCCAGTGTCGAATCTGCATGCGTAACTTCAGTCAGTCCTCCGACCTGTCCCGCCACATCCGCACCCACACCGGCGAGAAGCCTTTTGCCTGTGACATTTGTGGGAGGAAATTTGCCCGCTCCGACGCCCTGACCCAGCATACCAAGATACACACGCATCCCAGGGCACCTATTCCCAAGCCCTTCCAGTGTCGAATCTGCATGCGTAAGTTTGCCGACCGCTCCGACCTGTCCCGCCATACCAAGATACACACGGGCGAGAAGCCCTTCCAGTGTCGAATCTGCATGCGTAACTTCAGTCGCTCCGACAACCTGTCCGTGCACATCCGCACCCACACCGGCGAGAAGCCTTTTGCCTGTGACATTTGTGGGAGGAAATTTGCCGACCGCTCCAACCTGACCCGCCATACCAAGATACACCTGCGGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGAAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACAAAACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATC


4

GGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCTGATAA


5

Supplementary figures

Supplementary Figure 1

DSB

Deletion/insertion

NHEJHR

SSM

HomologyTemplate

SSM

DSB

GCCATCAGCCCTGTTCTTGGACTGTCAGCTGGT

CGGTAGTCGGGACAAGAACCTGACAGTCGACCA

FokI

FokI

5’

5’

DSB

Deletion/insertion

NHEJHR

SSM

HomologyTemplate

SSM

DSB

GCCATCAGCCCTGTTCTTGGACTGTCAGCTGGT

CGGTAGTCGGGACAAGAACCTGACAGTCGACCA

FokI

FokI

5’

5’


6

Supplementary Figure 1 ZFN-mediated DSBs repaired by either NHEJ or HR

pathways. The Mdr1a target site sequence is shown to be recognized by a pair of

ZFNs containing four and five fingers, respectively. The FokI DNase domain

dimerizes at the spacer between the binding sites and generates a DSB. When the

DSB is repaired via NHEJ, variable deletions or insertions are frequently introduced.

HR uses a homologous template to repair a DSB. If a donor with a sequence-specific

modification (SSM) flanked by homology is provided, the SSM is accurately

introduced into the targeted locus.


7


a

b

ATGAGGAAGATGGAGGTCTTCAAATCTGCCGTGTATGTGGGGACAAGGCCAATGGCTATCACTT CAATGTCATGACCTGTGAAGGAWT:

ATGAGGAAGATGGAGGTCTTCAAATCTGCCGTGTA GCGGCCGC GACAAGGCCAATGGCTATCACTTCAATGTCATGACCTGTGAAGNotI:

WT: GGAAGATGGAGGTCTTCAAATCTGCCGTGTATGTGGG --GACAAGGCCAATGGCTATCACTTCAATGTCATGACC 10/14

NotI: GGAAGATGGAGGTCTTCAAATCTGCCGTGTA GCGGCCGC GACAAGGCCAATGGCTATCACTTCAATGTCATGACC 4/14

Rat PXR

ATGAGGAAGATGGAGGTCTTCAAATCTGCCGTGTATGTGGGGACAAGGCCAATGGCTATCACTT CAATGTCATGACCTGTGAAGGAWT: ATGAGGAAGATGGAGGTCTTCAAATCTGCCGTGTATGTGGGGACAAGGCCAATGGCTATCACTT CAATGTCATGACCTGTGAAGGAATGAGGAAGATGGAGGTCTTCAAATCTGCCGTGTATGTGGGGACAAGGCCAATGGCTATCACTT CAATGTCATGACCTGTGAAGGAWT:

ATGAGGAAGATGGAGGTCTTCAAATCTGCCGTGTA GCGGCCGC GACAAGGCCAATGGCTATCACTTCAATGTCATGACCTGTGAAGNotI:

ATGAGGAAGATGGAGGTCTTCAAATCTGCCGTGTA GCGGCCGC GACAAGGCCAATGGCTATCACTTCAATGTCATGACCTGTGAAGATGAGGAAGATGGAGGTCTTCAAATCTGCCGTGTA GCGGCCGC GACAAGGCCAATGGCTATCACTTCAATGTCATGACCTGTGAAGNotI:





Rat PXR

Rat Mdr1a

NotI:TAACTCTTGTGATTTTGGCCATCAGCCCT GCGGCCGC GGACTGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGCCCGTGAGTC

WT:TAACTCTTGTGATTTTGGCCATCAGCCCTGTTCTTGGACTGTCAGCTGGTATTTGGGCAAAGGT AGGTGAAGCCCGTGAGTCCA

∆21+2:TAACTCTTGTGATTTTGGATTGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGCCCGTGAGTCC AGATTTTAAACTCTTTCTCAT

WT: TAACTCTTGTGATTTTGGCCATCAGCCCTGTTCTT --GGACTGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGC 0/31

NotI: TAACTCTTGTGATTTTGGCCATCAGCCCT GCGGCCGC GGACTGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGC 13/31

∆21+2: TAACTCTTGTGATTTTGG -------- AT------------- TGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGC 18/31

Rat Mdr1a







Rat Mdr1a


NotI:TAACTCTTGTGATTTTGGCCATCAGCCCT GCGGCCGC GGACTGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGCCCGTGAGTCTAACTCTTGTGATTTTGGCCATCAGCCCT GCGGCCGC GGACTGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGCCCGTGAGTC


WT:TAACTCTTGTGATTTTGGCCATCAGCCCTGTTCTTGGACTGTCAGCTGGTATTTGGGCAAAGGT AGGTGAAGCCCGTGAGTCCATAACTCTTGTGATTTTGGCCATCAGCCCTGTTCTTGGACTGTCAGCTGGTATTTGGGCAAAGGT AGGTGAAGCCCGTGAGTCCA


∆21+2:TAACTCTTGTGATTTTGGATTGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGCCCGTGAGTCC AGATTTTAAACTCTTTCTCATTAACTCTTGTGATTTTGGATTGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGCCCGTGAGTCC AGATTTTAAACTCTTTCTCAT





8

1 2 3 4 5 6 7 8

Kb

Rat PXR gene

2.0

1.5

1.0

Fetuses:

2.0

1.0

1 2 3 4 5 6 7 8

Kb

Rat Mdr1a gene

9 10 11 12 13 14Fetuses: 15

1.5

2.0

1.0

0.5

1 2 3 4

Kb

Mouse Mdr1a gene

Fetuses:1 2 3 4 5 6 7 8

Kb

Rat PXR gene

2.0

1.5

1.0

Fetuses: 1 2 3 4 5 6 7 8

Kb

Rat PXR gene

2.02.0

1.51.5

1.01.0

Fetuses:

2.0

1.0

1 2 3 4 5 6 7 8

Kb

Rat Mdr1a gene

9 10 11 12 13 14Fetuses: 15

1.52.02.0

1.01.0

1 2 3 4 5 6 7 8

Kb

Rat Mdr1a gene

9 10 11 12 13 14Fetuses: 15

1.51.5

2.0

1.0

0.5

1 2 3 4

Kb

Mouse Mdr1a gene

Fetuses:

2.02.0

1.01.0

0.50.5

1 2 3 4

Kb

Mouse Mdr1a gene

Fetuses:

d

TTGTGATTTTGGCCATCAGCTGGTATTTGGGCAAAGGTAGGTGAAGTCCATGAGTCCAGATTTT AAACTCTTTCC∆20:

∆48:TTGTGATTTTGGCCATCAGCCATGAGTCCAGATTTTAAACTCTTTCCCATGACCCACAGAAAAG TCTTTTAACCA

NotI:TTGTGATTTTGGCCATCAGCCCT GCGGCCGC GGACTGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGTCCATGA

WT:TTGTGATTTTGGCCATCAGCCCTGTTCTTGGACTGTCAGCTGGTATTTGGGCAAAGGTAGGTGA AGTCCATGA

WT: TTGTGATTTTGGCCATCAGCCCTGTTCTT --GGACTGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGTCCATGA 0

NotI : TTGTGATTTTGGCCATCAGCCCT GCGGCCGC GGACTGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGTCCATGA 17/47

∆48: TTGTGATTTTGGCCATCAGC -------------------------------------------------- CATGA 18/47

∆20: TTGTGATTTTGGCCATCAGC ---------------------- TGGTATTTGGGCAAAGGTAGGTGAAGTCCATGA 12/47

Mouse Mdr1a










TTGTGATTTTGGCCATCAGCTGGTATTTGGGCAAAGGTAGGTGAAGTCCATGAGTCCAGATTTT AAACTCTTTCCTTGTGATTTTGGCCATCAGCTGGTATTTGGGCAAAGGTAGGTGAAGTCCATGAGTCCAGATTTT AAACTCTTTCC∆20:


∆48:TTGTGATTTTGGCCATCAGCCATGAGTCCAGATTTTAAACTCTTTCCCATGACCCACAGAAAAG TCTTTTAACCATTGTGATTTTGGCCATCAGCCATGAGTCCAGATTTTAAACTCTTTCCCATGACCCACAGAAAAG TCTTTTAACCA


NotI:TTGTGATTTTGGCCATCAGCCCT GCGGCCGC GGACTGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGTCCATGATTGTGATTTTGGCCATCAGCCCT GCGGCCGC GGACTGTCAGCTGGTATTTGGGCAAAGGTAGGTGAAGTCCATGA


WT:TTGTGATTTTGGCCATCAGCCCTGTTCTTGGACTGTCAGCTGGTATTTGGGCAAAGGTAGGTGA AGTCCATGATTGTGATTTTGGCCATCAGCCCTGTTCTTGGACTGTCAGCTGGTATTTGGGCAAAGGTAGGTGA AGTCCATGA





Mouse Mdr1ac


9

Supplementary Figure 2 Sequence confirmation of NotI integration. The PCR

products of integration-positive fetuses in Figure 1 were cloned with TOPO TA

Cloning Kit (Invitrogen) following manufacturer’s instructions, and individual clones

were sequenced. In addition to the allele carrying NotI insertion, small deletions

resulted from NHEJ were also identified in the same fetus in some cases. All alleles in

each fetus are aligned to the wild type, with the number of each sequence found

among the total number of clones with readable sequences shown to the right. a Rat

Mdr1a locus, fetus #4, which has one NotI integrated allele and one allele resulting

from NHEJ with a 21 bp deletion and a 2 bp insertion. b Rat PXR locus, fetus #8,

which has one wild-type allele and one NotI integrated allele. c Mouse Mdr1a locus,

fetus #4, which has two different deletion alleles in addition to the NotI insertion, 20

bp and 48 bp, respectively. d PCR reactions corresponding to NotI digests in Figure 1

were resolved on 1% agarose gel, demonstrating that the smaller bands in each sample

positive for integration are in fact from NotI digestion.


10


a

1.5

0.5

1.0

2.0

M 1 7 2 3 4 5 6 M 1 7 2 3 4 5 6

b

1.5 1.0

2.0

0.5

M 1 7 2 3 4 5 6

c

TGGAAGCTAACTCTTGTGATTTTGGCCATCAGCCCTGTTCTTGGACTGTCAGCTGGTATTTGGG CAAAGGTAGGTGAAGCC

wt:

ATTTTGGCCATCAGCCCT GCGGCCGCAAGCTTAGGCCTCTGCAGTCTAGAGCGGCCGC GGACTGTCAGCTGGTATTTGGGCMCS:

TGGAAGCTAACTCTTGTGATTTTGGCCATCAGCCCTGTCAGCTGGTATTTGGGCAAAGGTAGGT GAAGCCCGTGAGTCCAGA

11 bpdeletion:

wt: TTTGGCCATCAGCCCTG ---------------------------------- TTCTTGGACTGTCAGCTGGTATTTGGGCAA 0/22

MCS: TTTGGCCATCAGCCCT GCGGCCGCAAGCTTAGGCCTCTGCAGTCTAGAGCGGCCGC GGACTGTCAGCTGGTATTTGGGCAA 10/22

Deletion: TTTGGCCATCAGCCCTG ---------------------------------- ----------- TCAGCTGGTATTTGGGCAA 12/22

NotI HIII NotIStuI PstI XbaI

TGGAAGCTAACTCTTGTGATTTTGGCCATCAGCCCTGTTCTTGGACTGTCAGCTGGTATTTGGG CAAAGGTAGGTGAAGCC

wt:

ATTTTGGCCATCAGCCCT GCGGCCGCAAGCTTAGGCCTCTGCAGTCTAGAGCGGCCGC GGACTGTCAGCTGGTATTTGGGCMCS:

TGGAAGCTAACTCTTGTGATTTTGGCCATCAGCCCTGTCAGCTGGTATTTGGGCAAAGGTAGGT GAAGCCCGTGAGTCCAGA

11 bpdeletion:

wt: TTTGGCCATCAGCCCTG ---------------------------------- TTCTTGGACTGTCAGCTGGTATTTGGGCAA 0/22

MCS: TTTGGCCATCAGCCCT GCGGCCGCAAGCTTAGGCCTCTGCAGTCTAGAGCGGCCGC GGACTGTCAGCTGGTATTTGGGCAA 10/22

Deletion: TTTGGCCATCAGCCCTG ---------------------------------- ----------- TCAGCTGGTATTTGGGCAA 12/22

NotI HIII NotIStuI PstI XbaI


11

Supplementary Figure 3 Targeted integration of a multiple cloning site (MCS) to the

Mdr1a locus in Long Evans Hooded (LEH) rats. The donor used here is slightly

different from that in Figure 1. Instead of a single NotI site, HindIII, StuI, PstI, and

XbaI sites were inserted into the NotI site using a pair of complimentary oligos. a

Genomic DNA from the toes were amplified with rat Mdr1a F and R primers, and the

PCR reactions were digested with restriction enzyme NotI. The founder lane is

indicated with an arrow head. b PCR reactions before NotI digestion. c Sequence of

the alleles. The PCR reaction for the founder was cloned, and 22 individual clones

were sequenced. Two different alleles were identified, one with the MCS integration

(in red), and the other with an 11 bp deletion (blue dashes). The number of clones

found containing each sequence is marked to the right of the sequence. No wild-type

sequence was detected in the sample.


12


b

a

M 1 62 3 5413 14 1918171615 121110987

Set 2) F+R

2.01.00.5

1 62 3 5413 14 1918171615 121110987

Set 3) F+GF

M

2.5

1.00.5

1 62 3 5413 14 1918171615 121110987

Set 4) R+GR

M

2.5

1.0

1 62 3 5413 14 1918171615 121110987

Set 1) GF+GR

M

1.5

0.5

M 1 62 3 5413 14 1918171615 121110987

Set 2) F+R

2.01.00.5

M 1 62 3 5413 14 1918171615 121110987

Set 2) F+R

M 1 62 3 5413 14 1918171615 121110987

Set 2) F+R

M 1 62 3 5413 14 1918171615 121110987

Set 2) F+R

2.01.00.5

2.01.00.5

1 62 3 5413 14 1918171615 121110987

Set 3) F+GF

M

2.5

1.00.5

1 62 3 5413 14 1918171615 121110987

Set 3) F+GF

M 1 62 3 5413 14 1918171615 121110987

Set 3) F+GF

M 1 62 3 5413 14 1918171615 121110987

Set 3) F+GF

1 62 3 5413 14 1918171615 121110987

Set 3) F+GF

M

2.5

1.00.5

1 62 3 5413 14 1918171615 121110987

Set 4) R+GR

M

2.5

1.0

1 62 3 5413 14 1918171615 121110987

Set 4) R+GR

M 1 62 3 5413 14 1918171615 121110987

Set 4) R+GR

M 1 62 3 5413 14 1918171615 121110987

Set 4) R+GR

M

2.5

1.0

1 62 3 5413 14 1918171615 121110987

Set 1) GF+GR

M

1.5

0.5

1 62 3 5413 14 1918171615 121110987

Set 1) GF+GR

M 1 62 3 5413 14 1918171615 121110987

Set 1) GF+GR

M 1 62 3 5413 14 1918171615 121110987

Set 1) GF+GR

M

1.5

0.5

1.5

0.5

0.5

1.52.5

1 2 53 4 6 7 8 9 101 2 53 4 6 7 8 9 10

Set 1) GF+GR Set 2) F+R

M

0.5

1.52.5

1 2 53 4 6 7 8 9 101 2 53 4 6 7 8 9 10


M 1 2 53 4 6 7 8 9 101 2 53 4 6 7 8 9 10


M

1 2 53 4 6 7 8 9 10

Set 3) F+GF

M

1.0

1.5

2.5

1 2 53 4 6 7 8 9 10

Set 3) F+GF

M 1 2 53 4 6 7 8 9 10

Set 3) F+GF

1 2 53 4 6 7 8 9 10

Set 3) F+GF

M

1.0

1.5

2.5

1 2 53 4 6 7 8 9 10

Set 4) GR+R

M

0.5

1.52.5

1 2 53 4 6 7 8 9 10

Set 4) GR+R

M 1 2 53 4 6 7 8 9 10

Set 4) GR+R

M 1 2 53 4 6 7 8 9 10

Set 4) GR+R

M

0.5

1.52.5


13

Supplementary Figure 4 PCR analysis on live born rats from embryos co-injected

with PXR or Mdr1a ZFN mRNA and respective donor plasmids. Tail clips were taken

from 7-day old pups. As in Fig. 2a, each sample was amplified in four sets of PCR

reactions with following primers: set 1) GF+GR; set 2) F+R; set 3) F+GF; and set 4)

R+GR. Primer set GF+GR amplifies GFP cassette (1.5 kb) from either targeted or

random integration. Set F+R primarily amplifies wild-type and NHEJ alleles. The

amplicon from the integrant is 1.5 kb larger in size and is rarely amplified efficiently

in the presence of other alleles. Junction PCRs with sets F+GF and R+GR amplify

only targeted integrant, yielding a product around 2.4 kb in size. a Rat PXR locus. The

GFP cassette correctly integrated into one (#4) of 10 pups. F+GF reaction was slightly

positive in all pups, likely to be nonspecific amplification since the amplicon from

pup #4 was the only sample yielded a readable sequence. b Rat Mdr1a locus. The

GFP cassette correctly integrated into one (pup #3) of 19 pups. Pup #19 also had

amplification of the GFP cassette but neither of the junctions, indicating a random

integration event.


14


a

b

bp1 3 4 5 6 7 8 9 102M

150

300

500

bp1 3 4 5 6 7 8 9 102M

150

300

500

1 62 3 5413 14 1918171615 121110987M

150

300

500

50

1 62 3 5413 14 1918171615 121110987M

150150

300300

500500

5050

3 10 13 16 17M

0.5

1.0

1.52.0

wild type

3 10 13 16 17M

0.5

1.0

1.52.0

wild type

Mdr1a

PXR


15

Supplementary Figure 5 NHEJ events in PXR and Mdr1a pups. a Large deletions in

Mdr1a pups. F+R samples containing bands that were significantly smaller than wild

type in Supplementary Figure 4b were rerun here, and the smaller bands were

isolated and purified for sequencing. Sequencing revealed various deletion sizes in the

following pups: #3: 513 bp and 6 bp; #10: 454 bp; #13: 602bp; #16: 344 bp; #17: 424

bp. The middle band in the lane for pup #3 turned out to be a mixture of the 6 bp and

513 bp deletions, likely a heteroduplex formed in later cycles of the PCR reaction.

PXR pups did not have large deletions. b Small deletions in PXR and Mdr1a animals

detected using mutation detection assay (see Methods). If both wild-type allele and

one or more mutated alleles containing NHEJ-induced deletions or insertions exist in

the same animal, mismatch will form at the target site after the denaturing and

reannealing reactions, which is cleaved by the nuclease to produce two smaller bands

in the gel. Thus, cleavage indicates presence of NHEJ-mediated deletions or

insertions (arrows). The allele with GFP insertion was not amplified efficiently

because of increased size. At the PXR locus, expected uncut PCR product was 357 bp,

and digested products were 160 bp and 197 bp. Pup #9 clearly was modified. Pup #4,

which carried the GFP integration, amplified a smaller band (35 bp deletion) without

cleavage, indicating the absence of wild-type allele. At the Mdr1a locus, PCR

amplified a 373 bp product. The cleaved bands were 227 bp and 146 bp. Four pups

were positive for NHEJ-mediated modifications (arrow). In summary, Two PXR pups

were modified by NHEJ (20%), one by HR (10%), and seven Mdr1a pups were

modified by NHEJ (37%), one by HR (5.3%).


16


2 3 4 5 6 7 8 9 10 11 1225 26 27 28 29 30 31 321 36353433

Set 3) F+GR

0.5

1.0

2.5

2 3 4 5 6 7 8 9 10 11 1225 26 27 28 29 30 31 321 363534332 3 4 5 6 7 8 9 10 11 1225 26 27 28 29 30 31 321 36353433

Set 3) F+GR

0.5

1.0

2.5

13 2014 19181716153837 242322214039

Set 3) F+GR

0.5

1.0

2.5

13 2014 19181716153837 24232221403913 2014 19181716153837 242322214039

Set 3) F+GR

0.5

1.0

2.5

2 3 4 5 6 7 8 9 10 11 1225 26 27 28 29 30 31 321 36353433Set 4) R+GF

0.5

1.0

2.5

2 3 4 5 6 7 8 9 10 11 1225 26 27 28 29 30 31 321 363534332 3 4 5 6 7 8 9 10 11 1225 26 27 28 29 30 31 321 36353433Set 4) R+GF

0.5

1.0

2.5

13 2014 19181716153837 242322214039

Set 4) R+GF

0.51.02.5

13 2014 19181716153837 24232221403913 2014 19181716153837 242322214039

Set 4) R+GF

0.51.02.5


17

Supplementary Figure 6 PCR analysis of the GFP integration into the mouse Mdr1a

gene. The GFP cassette was inserted into the donor in the same orientation of the

Mdr1a transcription. To detect the insertion junctions, primer F was paired with

primer GR, and primer R was paired with GF to amplify genomic DNA from 12.5-

day fetuses. Fetus IDs, 1 to 40, are labeled directly above each lane, with primer sets

above each gel. The samples were loaded in multichannel format. Expected amplicons

are about 2.4 kb (Supplementary Table 2). Fetuses #39 and 40 had both insertion

junctions. Fetus #16 only had one correct junction amplified, indicating a partial

integration. All junction amplifications were sequence confirmed. Fetus #6 has a band

amplified with F+GR. However, it is much smaller than expected. Sequencing shows

that it contains 300 bp from the PGK promoter of the GFP insertion as well as 200 bp

deletion from the right homology arm (not shown). Fetus #7 has a weak band similar

to that of #6 with primer set 4). However, we were not able obtain readable sequence

from the PCR product and could only conclude that #7, like #6, does not have a

correct integration.


18


a

b

1 2 6 7 8 9 10 11 124 53

2) 1) 2) 1) 2) 1) 2) 1) 2) 1) 2) 1) 2) 1) 2) 1)2) 1) 2) 1) 2) 1) 2) 1)M M

0.5

3.01.51.0

1 2 6 7 8 9 10 11 124 53

2) 1) 2) 1) 2) 1) 2) 1) 2) 1) 2) 1) 2) 1) 2) 1)2) 1) 2) 1) 2) 1) 2) 1)

1 2 6 7 8 9 10 11 124 53

2) 1) 2) 1) 2) 1) 2) 1) 2) 1) 2) 1) 2) 1) 2) 1)2) 1) 2) 1) 2) 1) 2) 1)M M

0.5

3.01.51.0

1 2 6 7 8 9 10 11 124 53

3) 4) 3) 4) 3) 4) 3) 4) 3) 4) 3) 4) 3) 4) 3) 4)3) 4) 3) 4) 3) 4) 3) 4)M M

0.5

2.5

1.0

1 2 6 7 8 9 10 11 124 53

3) 4) 3) 4) 3) 4) 3) 4) 3) 4) 3) 4) 3) 4) 3) 4)3) 4) 3) 4) 3) 4) 3) 4)

1 2 6 7 8 9 10 11 124 53

3) 4) 3) 4) 3) 4) 3) 4) 3) 4) 3) 4) 3) 4) 3) 4)3) 4) 3) 4) 3) 4) 3) 4)M M

0.5

2.5

1.0

1 2462 3 5413 14 1918171615 121110987 23222120

Set 2) F+R

2.0

1.00.5

M 1 2462 3 5413 14 1918171615 121110987 232221201 2462 3 5413 14 1918171615 121110987 23222120

Set 2) F+R

2.0

1.00.5

M

1 2462 3 5413 14 1918171615 121110987 23222120

Set 1) GF+GR

3.01.51.00.5

M 1 2462 3 5413 14 1918171615 121110987 232221201 2462 3 5413 14 1918171615 121110987 23222120

Set 1) GF+GR

3.01.51.00.5

M

1 2462 3 5413 14 1918171615 121110987 23222120

Set 4) R+GR

2.5

1.0

0.5

M 1 2462 3 5413 14 1918171615 121110987 232221201 2462 3 5413 14 1918171615 121110987 23222120

Set 4) R+GR

2.5

1.0

0.5

M

0.5

1.0

2.51.5

25 +26 27

Set 2) F+R

25 +26 27

Set 1) GF+GR

25 +26 27

Set 4) R+GR

M

0.5

1.0

2.51.5

0.5

1.0

2.51.5

25 +26 27

Set 2) F+R

25 +26 27

Set 1) GF+GR

25 +26 27

Set 4) R+GR

M 25 +26 27

Set 2) F+R

25 +26 27

Set 1) GF+GR

25 +26 27

Set 4) R+GR

M


19

Supplementary Figure 7 Examples of PCR identification of F1 animals carrying the

targeted integration allele. a Litter #2 of the F1 generation of Mdr1a founder #3. DNA

from tail clips of the pups (labeled as 1 to 12) was PCR amplified with all four sets of

primers, which are labeled above each lane. Pups #2, 5, 6, 10, and 12 had

amplification from all sets and had inherited a copy of the targeted integration allele.

b Litters #2 and 3 of the F1 generation from PXR founder #4. The pups were

numbered 1-27 for labeling convenience. Primer sets 1, 2 and 4 were used and labeled

above respective gels along with pup ID numbers. The samples were loaded in

multichannel format. Pups 2, 3, 7, 9, 10, 11, 13, 15, 19, 20, 21, 22, 23, and 24 had

amplifications in all three reactions and inherited a copy of the targeted integration

allele. Pup #1 did not have amplification in any of the reactions, and it was not

analyzed further and excluded from Supplementary Table 4.


20


Supplementary Figure 8 Additional founders at each site. a Founder #4-5. The

Mdr1a probe (on the right) and the GFP probe were hybridized sequentially. wt1 and

wt2 were from two wild-type genomic preps. As in Figure 2c, genomic DNA was

digested with restriction enzyme PciI. The expected fragment from wild-type allele is

3.0 kb, and integration allele, 4.5 kb. Founder #4-5 carried an allele with 147 bp

deletion resulted from NHEJ, which correlates to the band slightly below 3.0 kb. b

Five pups from the second injection session were analyzed by PvuII digestion

followed by hybridizing to PXR and GFP probes, sequentially.

a

b

2.02.3

9.46.5

4.3

23

M 2-1 2-2 2-3 2-4 2-5

GFP

6.2

2-1 2-2 2-3 2-4 2-5

PXR

6.2 4.7

2.02.3

9.46.5

4.3

23

M 2-1 2-2 2-3 2-4 2-5

GFP

6.2

2-1 2-2 2-3 2-4 2-5

PXR

6.2 4.7

2.02.3

23

9.46.54.3 4.5 kb

3.0 kb

∆147 bp

GFP Mdr1a

wt wt 4-5 wt wt 4-5M

2.02.3

23

9.46.54.3 4.5 kb

3.0 kb

∆147 bp

GFP Mdr1a

wt wt 4-5 wt wt 4-5M


21


Figure 2c Rat PXR/PXR probeFigure 2c Rat PXR/PXR probeFigure 2c Rat PXR/GFP probeFigure 2c Rat PXR/GFP probe

Figure 2c Rat Mdr1a/GFP probeFigure 2c Rat Mdr1a/GFP probe Figure 2c Rat Mdr1a/Mdr1a probeFigure 2c Rat Mdr1a/Mdr1a probe

Figure 3a Mdr1a F1/Mdr1a probeFigure 3a Mdr1a F1/Mdr1a probeFigure 3a Mdr1a F1/GFP probeFigure 3a Mdr1a F1/GFP probe


22

Figure 3c PXR F1/PXR probeFigure 3c PXR F1/PXR probeFigure 3c PXR F1/GFP probeFigure 3c PXR F1/GFP probe

Figure 3b Mdr1a F2/Mdr1a probeFigure 3b Mdr1a F2/Mdr1a probe


23

Supplementary Figure 9 Full-length pictures from which Figures 2c & 3 are

cropped. The portions used in figures are highlighted by boxes with panel names

indicated at the bottom of the pictures.


24

Supplementary Table 1 List of primers. Target Primer Sequence Genomic Coordinates

KpnI-F 5’-aaaaggtaccatgctgtgaagcagatacc chr 5: 8685459-8685477 NotI-R 5’-gtccgcggccgcagggctgatggccaaaatc chr 5: (-) 8686247-8686265 NotI-F 5’-cctgcggccgcggactgtcagctggtatttg chr 5: 8686272-8686291 SacII-

R 5’-aaaaccgcggctgaaaactgaatgagacatttgc chr 5: (-) 8687047-8687069

F 5'-tggcagctggaagacagata chr 5: 8685295-8685314 R 5'-cagaaccatttgcaactatgaca chr 5: (-) 8687133-8687155

Seq-F 5’-ctgtttcttgacaaaacaacactaggctc chr 5: 8686032-8686060

Mouse Mdr1a

Seq-R 5’-gggtcatgggaaagagtttaaatc chr 5: (-) 8686324-8686345 KpnI-F 5’-aaaaggtaccggagataggctggtttgacg chr 4: 21737712-21737731 NotI-R 5’-gtccgcggccgcagggctgatggccaaaatc chr 4: (-) 21738575-21738594 NotI-F 5'-ccctgcggccgcggactgtcagctggtatttg chr4: 21738601-21738620 SacII-

R 5’-aaaaccgcggatggtggtagttcggatgg chr4: (-) 21739299-21739317

F 5'-cgttgcctacatccaggtttc chr 4: 21737619-21737639 R 5'-agctgaaaatggaatttggtg chr 4: (-) 21739377-21739397

Seq-F 5’-ttggcaaaacaaaactggct chr 4: 21738371-21738390 Seq-R 5’-ttagcaaaaagcatgaaattgtg chr 4: (-) 21738721-21738743 Probe-

F 5'-atcccatgcaccagaaaatc chr 4: 21736801-21736820

Rat Mdr1a

Probe-R 5'-agcatcggtgattttggaag

chr 4: (-) 21737236-21737255

KpnI-F 5’-aaaaggtacctcagactggtccagattttagatttaaggg

chr 11: 64261093-64261122

NotI-R 5’-ttgtcgcggccgctacacggcagatttgaagacctc chr 11: (-) 64261831 64261853 NotI-F 5’-gtgtagcggccgcgacaaggccaatggctatcac chr 11: 64261861-64261880 SacII-

R 5’-aaaaccgcggataaatctactggttcgccaagctag chr 11: (-) 64262702-64262727

F 5’-tcagggaagagatggctctg chr 11: 64261020-64261039 R 5’-cagaggggactctgttctgg chr 11: (-) 64262814-64262833

Seq-F 5’-agcatctccctgaacaaacg chr 11: 64261660-64261679 Seq-R 5'-cattgtccagcaagttgacg chr 11: (-) 64261997-64262016 Probe-

F 5'-cattggcctagaagcattggg chr 11: 64263048-64263068

Rat PXR

Probe-R 5'-gtcacagtgttctatcccagc

chr 11: (-) 64263225-64263245

GF 5’-aaaagcggccgcttggggttgcgccttttcc GR 5’-aaaagcggccgccatagagcccaccgcatc

Probe-F 5'-tgcagagagcgacgagagc

GFP

Probe-R 5'-gtgcatgtggctgtccacca

Genomic coordinates on the rat and mouse genes are based on BLAT results from the UCSC Genome Bioinformatics site. The mouse genome assembly is July 2007 (NCBI37/mm9). The rat genome assembly is Nov. 2004 (Baylor 3.4/rn4). In primers containing restriction sites at the end, coordinates are only for the underlined gene specific nucleotides. (-) refers to minus strand. The residing chromosomes for the primers are also indicated.


25

Supplementary Table 2 Sizes of homologous arms in donors, NotI digested fragments and junction PCR products.

Target Mouse Mdr1a Rat Mdr1a Rat PXR F/R amplicon (bp) 1861 1779 1814

Primers KpnI-F, NotI-R KpnI-F, NotI-R KpnI-F, NotI-R Arm Length (bp) 808 883 761 NotI Digest (bp) 975 980 838

Left Arm

Junction PCR (bp) 2475 2480 2338 Primers Not-F, SacII-R Not-F, SacII-R Not-F, SacII-R

Arm Length (bp) 799 718 868 NotI Digest (bp) 888 801 978

Right Arm

Junction PCR (bp) 2388 2301 2478 HA-L: left homologous arm, amplified by primers KpnI-F and NotI-R HA-R: right homologous arm, amplified by primers NotI-F and SacII-R Expected size from NotI digest: the expected size from NotI digestion of each PCR product amplified with primers F and R Junction PCR: the expected size of each PCR product amplified with primers F or R paired with GFP-F or GFP-R at the GFP-integration junctions, which are the sum of the size of NotI-digest and 1500 bp of GFP cassette.


26

Supplementary Table 3 Genotype of all founders analyzed at the target site.

Target Donor Strain ID* No. of Alleles Genotype** Mdr1a NotI SD 4 2 TI, 21 bp deletion with 2 bp insertion PXR NotI SD 8 2 TI, wt

Mdr1a NotI FVB 4 3 TI, 20 bp deletion, 48 bp deletion Mdr1a GFP FVB 39 2 TI, wt Mdr1a GFP FVB 40 3 TI, 19 bp deletion, wt

Mdr1a GFP SD 3 3 TI, 513 bp deletion, 6 bp deletion

Mdr1a GFP SD 4-5 2 TI, 147 bp deletion PXR GFP SD 4 3 TI, 35 bp deletion, wt PXR GFP SD 2-1 2 TI, wt PXR GFP SD 2-2 2 TI, 236 bp deletion

Mdr1a MCS LEH 4 2 TI, 11 bp deletion NotI: Donor constructs with NotI site inserted between the homologous arms (Figure 2a) GFP: Donor constructs with GFP cassette inserted between the homologous arms (Figure 3a) MCS: Mdr1a donor construct with multiple cloning site inserted in the NotI site in NotI donor SD: Sprague Dawley rats; FVB, FVB mice; LEH: Long Evans Hooded rats * ID refers to the number a fetus or live-born pup was assigned. A single number is used on animals from the first round injection. For additional injections, the pup number is proceeded by a number that specifies the injection session with a dash. ** Genotype refers to the nature of the sequence at the target site: TI, allele resulted from targeted integration of NotI, GFP or MCS insertion; wt, wild-type allele; deletion/insertion: given number of base pairs being deleted or inserted at the target site.


27

Supplementary Table 4 Genotype of the F1 generation.

Founder Number of Litters

Total Animals TI 513 bp

deletion 6 bp

deletion Mdr1a #3 3 40 19 6 15

Founder Number of Litters

Total Animals

TI/Extra GFP locus

35 bp deletion Wt

PXR #4 4 57 29/6 26 2

The F1 generation carries one wild-type allele from the wild-type parent and one allele from the founder animal. All mutant alleles were identified by PCR followed by mutation detection assay and/or sequencing. Targeted integration alleles were further confirmed using Southern blots. TI: targeted integration. * The extra band hybridizing to GFP probe was present in PXR founder #4 (Fig. 2c), which is not at the targeted locus.


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