All animal procedures were in compliance with the institutional animal care guidelines and were approved by Stanford University's Administrative Panel on Laboratory Animal Care (A-PLAC, protocol number 14007).
Recombinant DNA was constructed using standard techniques. When fragments were amplified by PCR, we used Phusion Taq polymerase (Finnzymes), and confirmed the sequences fully by DNA sequencing. All synthetic DNA fragments were also fully confirmed by DNA sequencing.
pMADMα (pCA-FRT-G-Neo-T-FRT-T-Neo-G-FRT-pA). pCA
promoter (containing the chicken β-actin promoter and a CMV enhancer) and the SV40 polyadenylation signal (pA
) from pCA (HZ2)
, and synthetic DNA fragments containing FRT
sites were sequentially introduced into pBluescript to create a plasmid intermediate, pKM3
). The XmaI/EcoRI
fragment of MADM-TG
(see construction details below) was introduced into a pBluescript vector to flank this cassette with SpeI
. Then, the SpeI/HindIII
restriction fragment of MADM-TG
cassette, and XmaI/EcoRI
fragment of MADM-GT
were sequentially introduced into pKM3
to generate pMADMα
. The construct was digested with restriction enzymes PvuI and AflIII, and the insert was gel-purified using Qiagen gel extraction kit and eluted into 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA. The purified and linearized DNA contained ~50 bp and ~300 bp of vector sequence at its 5′ and 3′ ends, respectively. This vector sequence was deliberately retained to minimize the transgene damage with exonucleases after electroporation of the DNA into mouse ES cells.
pMADMβ (pFRT5-pCA-βGeo-pA-pPGK-TK-pA-FRT). The following fragments were assembled together in this order to make pMADMβ:
- 5′ protection arm: ~500 bp PCR fragment of β-lactamase gene from pBluescript amplified by the following primers: GGTACCATTTAAATAGATTATCAAAAAGGATCTTCACC and GGTACCTAACTCGCCTTGATCGTTGG.
- 319 bp PCR fragment of wheat germ agglutinin (WGA) gene amplified by PCR primers: GGTACCGACGTGTCCCAACAACCACT and AAGCTTCATGCCACAGGATCCCCACT. This arm was placed immediately downstream of the protection arm to provide unique sequence in the mouse genome for the Splinkerette PCR. Single NlaIII restriction site was artificially introduced in the 3′ end of this arm for Splinkerette PCR.
- FRT5 (GAAGTTCCTATTCCGAAGTTCCTATTCTTCAAAAGGTATAGGAACTTC) from a synthetic DNA was introduced after the unique 5′ arm.
- pCA promoter from pCA (HZ2)
- SalI/KpnI fragment containing the βGeo gene from the plasmid Z/EG
- KpnI/HindIII fragment containing the pPGK-TK-pA cassette from the plasmid pLOXPNT
. (Note that we eventually decided not to use thymidine kinase (TK)-based selection.)
- FRT (GAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC) from a synthetic DNA was introduced after the TK cassette.
- 218 bp PCR fragment of yeast His3 gene amplified by PCR primers GCGGCCGCTCGAAGTAGCCGCCGTTGTTGTTAT and GAGCTCGGTGATAGGTGGCAAGTGGT. (Note: This sequence was originally introduced as a 3′ unique area for screening purposes, but was not used in this study.)
The construct was digested with restriction enzymes PvuI and AflIII, and the insert was gel-purified using Qiagen gel extraction kit and eluted into 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA. The purified and linearized DNA contained ~500 bp and ~3000 bp of vector sequence at its 5′ and 3′ ends, respectively. The vector sequences served as protection arms against endonucleases after electroporation into the mouse ES cells.
and pExT (pFRT5-pCA-GT-pA-FRT-attPx3-pPGK-Hyg-pA-attB
: Synthetic DNA fragments containing FRT5
were sequentially introduced 5′ and 3′ to the MADM cassettes in the MADM-GT
constructs to generate intermediates pFRT5-GT-FRT
. Independently, pattB-Hyg-attPx3
was generated by flanking the hygromycin resistance gene (Hyg
) driven by the phosphoglyceratekinase promoter (pPGK
) with ϕC31 integrase recognition sites: three 70-bp long attP
sites from pBT298
and a “full length” attB
. To create the final constructs (pExG
), the XmaI/BamHI
fragment from pattB-Hyg-attPx3
was introduced into the SwaI
site of pFRT5-GT-FRT
. The constructs were prepared by using Endotoxin Free-Maxi prep (Qiagen) for the electroporation into the mouse ES cells.
To construct and test final MADM targeting constructs we created a set of constructs in the pCA (HZ2)
plasmid, which contains a polylinker between the pCA
promoter (chicken β-actin promoter and CMV enhancer) and an SV40
polyadenylation site 
The previously used first GFP
exon from the GR
were assembled in pHZ2
separated by the previously described modified β-globin intron
The previously used second GFP exon from the RG
was assembled with a fragment containing a Kozak sequence, ATG start codon and the same β-globin intron
desribed above in pHZ2
pCA-G-intronNeo-T and pCA-T-intronNeo-G.
We inserted a BglII/BamHI
fragment containing the neomycin resistance gene (Neo
) driven by an SV40
promoter and followed by the HSV TK
polyadenylation site into the BglII
site of pCA-G-intron-T
, respectively. We created three different versions of the Neo
cassette to contain different numbers or identities of recombination sites: version 1: pLN: loxP-pSV40-Neo-pA; version 2:pFLN: FRT-loxP-pSV40-Neo-pA; version 3: pFLLFLN: FRT-Lox5171-Lox2272-FRT-loxP-pSV40-Neo-pA
. The loxP
are incompatible with each other and with loxP
, but each one is compatible with itself 
. They were introduced in attempts to increase recombination efficiency. Comparisons of these intron versions and their effect on recombination efficiency will be described elsewhere (A. Henner and H. Zong, in preparation). The intron versions that were used for creating targeting constructs for particular loci described in this study are schematically represented in Figure S1
pBT234 (pCA-G-intron-tTA2ATG-less-pA). Used for testing the cassettes before the construction of final targeting constructs.
pBT250 (pCA-G-intronNeo-tTA2ATG-less-pA). pGLLFNL was cloned into BglII site of pBT234.
pBT270 (pCA-G-intronNeo-tTA2ATG-less-pA-ii-TRE-tdT3Myc-pA-ii). ii-TRE-tdT3Myc-pA-ii
was inserted into pBT250
All targeting constructs for the Rosa26
locus were created by inserting a PmeI/AscI
-digested fragment from a precursor plasmid into the pROSA26-PA
. The pRosa26-GT
precursor is pCA-G-intronNeo-T
precursor is pCA-T-intronNeo-G; pRosa26-G-tTA2 (pBT259)
precursor is pBT250
; pRosa26-GTET (pBT272)
prescursor is pBT270
Used as a positive control for GFP expression. GFP4m (or mut4EGFP) is a thermotolerant GFP variant 
. All other GFP-containing constructs in this study contain this variant of GFP.
. Described originally in 
. Used as a positive control for split GFP expression.
pBT225 (pCA-tdT3Myc): Used as a positive control for tdT expression and Myc staining.
. Constructed initially as a test for splitting the tdT
gene into an exon containing a start codon (ATG
) and and an exon containing the rest of tdT by the β-globin intron
containing the BglII
site and loxP
Used as a positive control for tTA2 activity in conjunction with pBT239
gene was subcloned from pUHT61-1
Constructed initially as a test for splitting the tTA2
gene into ATG
and the rest of tTA2
by the β-globin intron
. PCR was used to construct two DNA fragments: XmaI-ATG-intron
. The β-globin intron
was modified at the same position as described previously 
to contain single BglII
sites. The two fragments were ligated to each other (via blunt ends) and to BglII/EcoRI
-digested pCA (HZ2)
in a 3-way ligation reaction. This construct was used to test the functionality of tTA2 after insertion of the intron in conjunction with pBT239
pBT267 (pCA-ATG-intron-tTA2ATG-less-TRE-tdT3Myc-pA) and pBT268 (pCA-ATG-intron-tTA2ATG-less-iiTRE-tdT3Myc-pAii). Constructed to compare the effect of insulators on decreasing tTA2-independent activation of TRE. The constructs were also tested in the presence of doxycycline to assess which construct has higher background expression of tdT3Myc.
Screening of ES cell clones obtained by random transgenesis
We used standard techniques 
to modify R1 mouse ES cells, which originated from a 129 mouse strain 
construct was introduced into ES cells via electroporation, and individual G418-resistant clones were evaluated for intact transgene integration by genomic PCR using primers KM1 (GTGCTGCAAGGCGATTAAGT
) and KM2 (TTATGTAACGCGGAACTCCA
) to detect the 5′ end of each transgene (PCR product, 211 bp), and CCCCCTGAACCTGAAACATA
to detect the 3′ end of each transgene (PCR product, 275 bp). We further analysed the genomic DNA from the ES cells containing intact transgenes by Southern blotting. We used a probe for Neo
, which is located in the intron of MADM cassette, and the genomic DNA obtained from the R26TG/+
mouse line as a reference for a single-copy transgene. For ES cells that contained single-copy transgenes based on the Southern blot, we performed inverse PCR to identify the 5′-flanking genome sequence. Genomic DNA was digested with restriction enzyme NlaIII and subjected to the ligase-mediated self-ligation. The resultant circular DNA was then used as a template for a two-step nested PCR to amplify the transgene flanking region. For the first round of PCR, we used primers: TAATCGAAACCCTGGCGTTA
. For the second round of PCR, we used 0.3 µl of the first round PCR product and primers: ACTTAATCGCCTTGCAGCAC
. The final PCR products were analyzed by electrophoresis, gel-extracted by the Qiagen gel extraction kit, and analyzed by DNA sequencing. For the intact single-copy transgenes integrated in intergenic regions close to any centromere, we performed additional Splikerette PCR 
to confirm integration sites.
was also introduced into R1 mouse ES cells by electroporation and individual G418-resistant clones in 96-well plates were evaluated for the expression of βgeo by lacZ staining. 96-well plates were washed with PBS, fixed by 0.2% glutaraldehyde in PBS for 5 min. at room temperature (RT), and washed 3 times at RT with the staining buffer (2 mM MgCl2, 0.01% Deoxycholate, 0.02% NP40, 100 mM phosphate buffer, pH 7.5). Cells were then treated with the solution containing: 5 mM potassium ferricyanide (Sigma), 5 mM potassium ferrocyanide (Sigma), 2.5 mM 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal, Invitrogen) at 37°C for 1-2 hours. We recorded the activity of lacZ in individual clones and then extracted genomic DNA from 96-well plates. We tested the intactness of 5′ and 3′ ends of the transgenes by PCR. We used the following primers: CTATGCCCGAACAACCTCTG
5′ end); CCCCCTGAACCTGAAACATA
3′ end). The clones with high lacZ expression and intact transgenes, were further analyzed by Splinkerette PCR 
to identify the flanking genomic sequence at the 5′ end of the transgene. Splinkerette PCR was reported to be more efficient than inverse PCR method we used for pMADMα
screening. The genomic DNA was digested with NlaIII and a specifically designed Splinker was ligated overnight at 16°C. To make the Splinker adaptor, two synthetic DNA oligonucleotides: TAACCGTTGCTAGGAGAGACCGTGGCTGAATGAGACTGGTGTCGACACTAGTGGCATG
were annealed. The ligation products were purified by the Qiagen PCR purification kit and eluted into 30 ml of TE. 1 µl of the sample was used for the first round of PCR with primers: AACCGTTGCTAGGAGAGACC
. 0.3 µl of the first PCR product was subjected to the second round of PCR with primers: GCTGAATGAGACTGGTGTCG
. The final PCR products were analyzed by electrophoresis, gel-extracted by the Qiagen gel extraction kit, and analyzed by DNA sequencing.
Converting pMADM transgenes into GT or TG cassettes
To convert pMADMα
, 5 µg of a plasmid (pPGK-Flpo-Puro
) containing a codon-optimized Flp recombinase gene (Flpo
and the puromycine resistance gene
was introduced into 5 million cells of selected clones via electroporation. We cultured ES cells with 200 µg/ml G418 and 2 µg/ml puromycine for 48 hours, and ES cell were subsequently cultured only in the presence of G418 until colonies formed. Individual G418-resistant clones were analyzed by two genomic PCRs to specifically detect 5′ and 3′ parts of pMADMα
and identify non-recombined clones and partially recombined clones (GT
cassette). One PCR used primers GCAACGTGCTGGTTATTGTG
to amplify ~600 bp product for non-recombined pMADMα
cassette and MADM-GT
cassette or ~300 bp product for MADM-TG
cassette. Another PCR used primers GAAACTGGGCATGTGGAGAC
to amplify ~800 bp product for non-recombined pMADMα
cassette and MADM-TG
cassette or ~2.0 kb PCR product for MADM-GT
cassette. The ES cell clones that contained correctly recombined cassettes were used to generate chimeric mice by injection into C57BL/6 blastocysts.
To convert pMADMβ'1
, 25 mg of one of the exchange cassette plasmids (ExG
) and 25 mg of pPGKFLPobpA
plasmid (Addgene plasmid 13793) 
were introduced into the selected ES cells (~5×106
cells). We cultured the ES cells without selection for 72 hours and then applied hygromycin (120 mg/ml) for one week. Individual hygromycin-resistant clones in 96-well plates were divided into five replicas: two for stock, one for lacZ staining, and two for genomic DNA preparation. To detect the FRT5
mediated site-specific recombination events, we used PCR with primers: CTATGCCCGAACAACCTCTG
to amplify the junction containing FRT5
. This PCR not only confirmed that the ES clone still contained the MADM transgene, but also generated different sizes of PCR products for non-recombined (510 bp) and recombined (550 bp) clones. The PCR products were analyzed by electrophoresis on a 2% agarose gel. Site-specific recombination was confirmed by additional PCR primer sets that specifically amplify the newly formed junctions at FRT5
(5′ of the transgene): CTATGCCCGAACAACCTCTG
(create a 286 bp product) and FRT
(3′ of the transgene): AAGCATCAACGACAACAACG
and 5′- CGGAATACCACTGAAATTGG
(create a 200 bp product). The ES cell clones that contained correctly recombined cassettes were used to generate chimeric mice by injection into C57BL/6 blastocysts. The chimeras were directly crossed to a ϕC31o integrase mouse line (Jackson laboratory, stock# 007670) 
to remove the pPGK-Hyg-pA
cassette from the genome in the next generation.
Tissue processing, immunohistochemistry and imaging
Tissues were processed according to previously described procedures 
. Neither tdT nor GFP required immunostaining for visualization. Although the majority of the data presented in the paper were obtained from unstained tissue sections, sections can be immunostained for better signal preservation according to previously published methods 
using the following primary antibodies: chicken anti-GFP (1
500; Aves Labs), goat anti-MYC (1
200; Novus; the best results are obtained if antibody is pre-absorbed with fixed, finely minced, wild-type brain according to the previously described procedure 
), rabbit anti-DsRed (1
1000; Clontech), or rabbit anti-LacZ (1
500; MP Biomedicals (previously Cappel) Cat. No. 0855976). Secondary antibodies (donkey anti-chicken FITC, donkey anti-rabbit Cy3 and donkey anti-goat Cy3 from Jackson ImmunoResearch), were used at 1
200 dilution. In some cases, sections were also stained with DAPI. Sections were imaged with a Nikon CCD camera or a confocal microscope (Zeiss 510).
Mouse DNA was extracted and genotyping PCR performed as described previously 
For genotyping M1 MADM transgenes, we used primers: KM5 (CTATGCCCGAACAACCTCTG), KM6 (ATCATATGCCAAGTACGCCC), KM7 (GGGGTCGATCTTGTCAGTCT) and KM8 (TTGCGTTGCAATTTTCTGAG). These primers amplify a 512 bp transgene fragment and a 700 bp wt M1 locus fragment.
For genotyping M10 MADM transgenes, we used primers: KM1 (GTGCTGCAAGGCGATTAAGT) and KM2 (TTATGTAACGCGGAACTCCA). These primers amplify a 211 bp fragment for either MADM cassette. To distinguish heterozygous vs. homozygous transgene, we used additional primers KM3 (CATATTCCAAAGCTACCACACACT) and KM4 (ATCATGGAGGAGCAGTGGAG), which amplify a 300 bp fragment from the wt M10 locus.
MADM transgenes (available at The Jackson Laboratory: MADM-11-GT, stock# 013749 and MADM-11-TG, stock# 013751) were genotyped as described 
, using primers: SH176 (TGGAGGAGGACAAACTGGTCAC
), SH177 (TCAATGGGCGGGGGTCGTT
), SH178 (TTCCCTTTCTGCTTCATCTTGC
) according to the genotyping protocol deposited to The Jackson Laboratory.
For genotyping Rosa26 knock-ins we used primers Rosa4 (TCAATGGGCGGGGGTCGTT), Rosa10 (CTCTGCTGCCTCCTGGCTTCT) and Rosa11 (CGAGGCGGATCACAAGCAATA). These primers amplify a 250 bp knock-in fragment and a 330 bp wt Rosa26 locus fragment.
For genotyping TRE-KZ, we used primers Tau1 (GGTGGCAAGGTGCAGATAAT) and Tau2 (CAGCTTGTGGGTTTCGATCT) to amplify a 315 bp tau fragment. We combined them with primers IMR0015 (CAAATGTTGCTTGTCTGGTG) and IMR0016 (GTCAGTCGAGTGCACAGTTT) to amplify a 200 bp internal control fragment.
For genotyping Foxg1tTA, we used primers Ling40 (TCTGCACCTTGGTGATCAAA) and Ling57 (ATCGCGATGGAGCAAAAGTA) to amplify a 270-bp fragment of tTA. We combined them with primers Globin1 (CCAATCTGCTCACACAGGATAGAGAGGGCAGG) and Globin2 (CCTTGAGGCTGTCCAAGTGATTCAGGCCATCG) to amplify a 500 bp internal control fragment.
For genotyping Cre transgenes, we used Foxg1-Cre-A (CACCCTGTTACGTATAGCCG) and Foxg1-Cre-B (GAGTCATCCTTAGCGCCGTA) to amplify a 300 bp transgene fragment and internal Globin primers as described for tTA genotyping to amplify the internal control.
Genotyping for the presence of the neomycin resistance gene (Neo) was performed using primers IMR3742 (GTGAGCTGCACTTCCAGAAG), IMR3743 (GACTTTCGGCATGTGAAATG), IMR013 (CTTGGGTGGAGAGGCTATTC) and IMR014 (AGGTGAGATGACAGGAGATC). These primers produce a 280 bp Neo band and a 180 bp wt band.
Mouse maintenance and crosses
All mice were kept in a mixed background. All mouse lines contained some 129 and CD1 strain backgrounds, and some additionally contained C57Bl/6 and FVB. We preferred to keep mice with as much CD1 background as possible to increase fecundity.
We kept GT and TG stocks separately from each other. This approach prevents mixing up the stocks and allowed us to use the same PCR for genotyping either stock using the common pairs of primers. Other transgenes were crossed into one of the MADM cassette alleles. Once a Nestin-Cre or HprtCre line is crossed into one of the MADM cassette strains, the loxP-flanked (floxed) Neo is removed in the germline. Therefore any double positive animal of this type will transmit to its progeny the MADM cassette with removed Neo. The MADM cassette alleles were then usually homozygosed during maintenance to obviate the need for genotyping for that allele. For example, to generate the experimental animals R26TG/GT;Nestin-Cre+/−;TRE-KZ+/−, we would create two lines: R26GT/GT and R26TG/TG;Nestin-Cre+/−;TRE-KZ+/−. The first line, as well as other MADM-cassette lines, were usually kept homozygous (no genotyping required). In the case of R26GT/GT only, some homozygous males show decreased fertility, so from time to time a homozygous female was crossed to a CD1 wt male, and after that the homozygous stock was reestablished by crossing heterozygous mice to each other. The second line was created by sequentially introducing Nestin-Cre and TRE-KZ transgenes into R26TG mice. After the triple-transgenic mice R26TG/+;Nestin-Cre+/−;TRE-KZ+/− were created, they were crossed to R26TG/TG to create R26TG/TG;Nestin-Cre+/−;TRE-KZ+/−. These mice were maintained by crossing to R26TG/TG homozygous stock and genotyping only for the presence of Cre and tau.
All GG and TT alleles were generated by Cre-mediated interchromosomal recombination and were detected by screening tail samples for expression of GFP or tdT under the fluorescence microscope. In addition, under UV light, these animals appeared uniformly green and red, respectively. All GG and TT alleles had lost the floxed Neo from the intron as confirmed by Neo PCR (see Genotyping).
is located on the X chromosome 
. If maximal level of recombination is desired, it is recommended to use males for phenotypic analysis (HprtCre/Y
as opposed HprtCre/+
). In females, due to the random X inactivation, only roughly half of the cells have the active HprtCre
The DNA constructs described in this paper will be deposited to Addgene. We will also deposit the following lines to The Jackson Laboratory; R26GT
(stock# 017912), R26TG
(stock# 017921), R26TT
(stock# 017922), R26G-tTA2
(stock# 017909), Miya10GT
(stock# 017923), and Miya10TG
(stock# 017932). Note that we have already deposited the following lines to The Jackson Laboratory: Rosa26GG
(also called MADM-GG
; stock# 006053) 
(also called MADM-11GT
; stock# 013749), and Hipp11TG
(also called, MADM-11TG
; stock# 013751) 
. Whereas the GT
mice can be used for MADM analysis of genes located on those specific chromosomes, GG
mice express high-level green or red fluorescence proteins globally and can be used, for example, as tissue donors in transplantation or chimeragenesis experiments.