Human Molecular Genetics, 2000, Vol. 9, No. 12 1745-1751
© 2000 Oxford University Press
A Cre-lox recombination system for the targeted integration of circular yeast artificial chromosomes into embryonic stem cells
Department of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
Received 11 February 2000; Revised and Accepted 29 May 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The ability to produce embryonic stem (ES) cell lines containing different yeast artificial chromosomes (YACs) integrated into the same location in the genome provides a system for comparing the bio-logical effects of YAC transgenes without the confounding influences of integration site and copy number. A targeting system was developed for the directed integration of circular YACs into mouse ES cells. The system combines Cre-lox recombination technology, specifically a positive-selection integration system, with circular YAC lipofection technology to achieve single copy targeted integration of a transgene. Three independent germline competent ES cell lines [lox-containing ES lines (designated LES)] were created that contain a ‘-neo–lox’ cassette integrated at different sites within the ES genome. A plasmid containing YAC vector sequences and a complementary ‘-neo–lox’ cassette was used to circularize two linear YACs containing genomic DNA from human chromosome 21. The circularized YACs were then targeted to the lox sites of the LES cell lines. Polymerase chain reaction and Southern analysis demonstrated that 21% (5 of 24) of lox-recombinants contain a full-length intact YAC. This system will make the study of YAC transgenic mice more reliable and reproducible, allowing the potential for direct comparison of different transgenes expressed from the same site within the genome.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Yeast artificial chromosome (YAC) transgenic technology allows large chromosomal regions, in the order of several hundred kilobases, to be inserted into the genome of mice. YAC transgenic technology has been employed to study many basic issues in genetics, including the development of mouse models of human disease. Several groups have introduced defined genomic regions of human chromosome 21 cloned in YACs into transgenic mice in attempts to correlate dosage imbalance of genes with specific Down syndrome features (1–4). A number of YACs containing human and mouse genes have been introduced into mice and some of these display tissue- and stage-specific expression (reviewed in refs 5,6). Many YAC transgenic mouse lines, however, do not express genes from the inserted YAC in an appropriate pattern, and these differences in expression can be seen among lines containing the same YAC (J. Gearhart, unpublished data). This is thought to result from the highly variable number of copies integrated into the genome and from position effects on gene expression due to random integration.
Cre-lox recombination is a method to introduce transgenes into specific, single, defined sites within the mammalian genome to create a genetically reproducible environment for the study of expression of introduced transgenes. Fukushige and Sauer (7) designed a positive-selection lox integration system that allows for the direct selection of lox-recombinants in mammalian cells. This system was designed to directly select Cre-mediated DNA integration at a lox target previously placed in the genome of cultured mammalian cells. We have created modified positive-selection lox vectors to place and test single, functional lox sites within the mouse embryonic stem (ES) cell genome. Several lox-containing ES cell lines (LES) with independently integrated lox sites have been characterized. Two different YACs have been circularized in yeast by homologous recombination of a modified positive-selection lox vector, and intact circular YACs (cYACs) have been successfully targeted to the lox sites within the LES lines. This system provides an efficient means of creating YAC transgenic ES cells, with 21% of the neomycin resistance (NeoR) gene colonies generated having intact YAC.
This system will be reproducible, allowing genes from large vectors such as YACs to be consistently expressed from a unique integration site in ES cells. All YACs derived from a similar vector will be amenable to circularization due to the nature of the design of the circularizing vector. Moreover, the Cre-lox recombination system is very efficient and will facilitate recombination and thus integration of the circular molecule in a reproducible manner. The system described here will increase the efficiency of the production and study of YAC transgenic ES and mouse lines, and thus our ability to study the biologic basis of many human diseases.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Creation of ES lines containing lox sites
To allow for the site-specific insertion of YACs into the mouse genome, a single lox site was introduced into the ES cell genome. The construction of pSF1Hyg involved several modifications of the vector pSF1, which contains a single lox site adjacent to the 3' portion of a ‘-neo cassette’ consisting of the entire coding region of the NeoR gene except for a start codon and promoter. Recombinants between the 3' ‘-neo cassette’ and a second vector containing a 5' ‘-neo cassette’ (promoter and start site for NeoR) can be selected by exposure to the neomycin analog G418. A 1.4 kb cassette containing the hygromycin resistance gene (HygR) under the control of the DNA Polymerase II promoter was isolated from the vector polIIHyB and cloned into pSF1 for use as a mammalian selection marker during the random integration event. Orientation was determined to ensure that transcription of HygR was in the opposite direction of the 3' ‘-neo cassette’ to avoid any influence on transcription of NeoR.
The ES lines J1 (courtesy of R. Jaenisch, Whitehead Institute, Cambridge, MA) and R1 (courtesy of J. Rossant, Samuel Lunenfeld Research Institute, Toronto, Ontario) were chosen for transfection based on high rates of chimerism following injection into mouse blastocysts. The vector pSF1Hyg was co-lipofected with the Cre-expressing vector pBS185, which expresses the Cre-recombinase gene under the control of a human cytomegalovirus (hCMV) promoter that is active in ES cells, to reduce the occurrence of head-to-tail concatemer lox integration sites. Positive transformants were selected in media supplemented with hygromycin B. The first of two transfections yielded 71 ‘LES1’ lines for analysis, and the second transfection yielded 62 ‘LES2’ lines.
Characterization of LES lines
Southern analysis.
To identify LES lines containing a single copy of pSF1Hyg and lacking the Cre-expressing vector pBS185, LES lines were screened by Southern analysis. Figure 1 illustrates the strategy used to determine single-copy integration of pSF1Hyg. Southern blots were also hybridized with a probe for the Cre gene to ensure that the pBS185 vector did not integrate into the ES genome. Of 41 LES lines screened for pBS185, 29 (71%) did not retain an integrated vector (data not shown). Lines that exhibited multiple copy integration of pSF1Hyg or the presence of pBS185 were excluded from future experiments. Of 133 LES lines screened by Southern analysis, 42 (32%) were positive for single-copy integration and absence of pBS185.
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Testing recombination efficiency of lox sites in LES lines.
Control transfections were performed to test recombination at the integrated lox sites in individual LES lines. LES lines were co-transfected with the vector pLC2, which contains one lox site adjacent to a promoter and start site for the NeoR gene, and the Cre-expressing vector pBS185. When the lox site from pLC2 recombined with the integrated lox site adjacent to the 3' ‘-neo cassette’ from pSF1Hyg, cells became NeoR (Fig. 2A–C). Five LES lines, of 24 tested, exhibited recombination at the introduced lox site. The efficiency of generating neomycin resistance colonies for each LES line tested was: 5.4 x 10–6 for LES1–5, 6.8 x 10–6 for LES2–4 and 5 x 10–7 for LES2–79.
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Genomic DNA was harvested from NeoR colonies produced from control transfections and tested by polymerase chain reactions and Southern analysis for presence of the recombined lox site. PCR primers were designed from the sequences of pLC2 and pSF1Hyg such that a product is formed only when the two lox sites have recombined properly (Fig. 2D). All colonies tested by PCR were positive. Southern blot hybridization of digested genomic DNA confirmed lox PCR results (data not shown). PCR products from several colonies of each line were sequenced for confirmation of recombination. All control colonies were PCR-positive for HIS3 and for HygR.
Karyotyping and fluorescence in situ hybridization (FISH).
Karyotype analysis of LES lines 1–5, 2–4 and 2–79 revealed a normal chromosomal number of 40, indicating that no chromosomal abnormalities occurred during passage of cells. FISH performed using pSF1Hyg as a probe determined the integration site of the vector in the ES genome to be chromosome 19 in LES1–5, chromosome 17 in LES2–4 and chromosome 19 in LES2–79. The vectors pSF1Hyg and pLC2 were differentially labeled and hybridized to metaphase preparations of control colonies of LES2–4. Co-localization of the two different colored signals on the same chromosome indicated co-integration at a single site (data not shown).
Production of LES transgenic mice.
The successful production of mice homozygous for the integrated vector ensures that no sequences essential for growth or development were interrupted by insertion of pSF1Hyg. LES lines with the desired vector integration patterns were injected into mouse blastocysts to produce transgenic mouse lines. All potential homozygotes were screened by PCR for HygR gene, by Southern blot analysis and by mating (data not shown). Homozygous LES1–5 mice were developmentally normal and fertile, indicating that no essential sequences were interrupted by insertion of pSF1Hyg.
Mapping YACs C4C10 and C14B2
Two non-chimeric human chromosome 21q22.1–22.3-specific YACs, C4C10 and C14B2, were obtained from the Washington University Human YAC library (8). Previous evidence indicated that the 250 kb YAC C4C10 contains the AML1 gene, which is located at 21q22.3 (9). The 450 kb YAC C14B2 was restriction enzyme mapped previously (10), and contains all or part of the genes AML1 and KCNE1, indicating that these YACs overlap in sequence. PCR amplification of different regions of AML1, KCNE1, the gene DSCR1 and of HSA21 markers in the region was performed to map the contents of the two YACs. C14B2 contains KCNE1, the gene DSCR1 and the entire 250 kb region of sequence within C4C10. Both YACs lack the AML1 distal promoter, a sequence essential for expression of distal transcripts of AML1, and C4C10 also lacks AML exon 1. Figure 3 indicates the PCR products detectable on each YAC.
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Circularization of linear YACs
We designed plasmid pLC2 to circularize YACs with a pYAC-4 backbone by homologous recombination with the YAC arms (8). Sequences chosen for homology were the ampicillin resistance gene (AmpR) gene and sequence from the plasmid pBR322, which was introduced into pLC2. The selectable marker HIS3 was introduced into pLC2 for selection in yeast after transformation. For transformation into yeast, pLC2 was linearized with NruI and ScaI, leaving free ends homologous to linear YAC arms (Fig. 4). YPH925 yeast containing either linear YAC C4C10 or C14B2 DNA was transformed with linear pLC2. Transformants were selected on SD8-HIS3 plates and replica plated onto SD8-URA3 plates. Successful transformants were those that grew on SD8-HIS3 but not on SD8-URA3.
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Transformation of C14B2 yeast yielded two colonies (of 33) that grew on SD8-HIS3 but not on SD8-URA3. High molecular weight (HMW) DNA from transformants was separated by pulsed-field gel electrophoresis (PFGE), and the gel was blotted and hybridized with the probes Alu, PY1, KCNE1 and AML1 exon 7. Signal from all probes used appeared only in the well in lanes with transformant DNA, indicating that the YAC was circularized (Fig. 5A). HMW DNA plugs were digested with EcoRI and BamHI and subjected to electrophoresis, blotted and hybridized with probes for AML1 exon 7 and Alu. All samples gave the same banding pattern with each probe, indicating that no rearrangements had taken place during transformation and circularization of the YAC (Fig. 5B). Transformation of C4C10 yeast with pLC2 yielded five colonies (of 16) that grew on SD8-HIS3 but not on SD8-URA3. Chromosome-sized HMW DNA plugs were subjected to PFGE and blotted, as above, and blots were hybridized with PY1. Signal was observed in the well only for all cYACs (Fig. 5A). A blot of NotI digests showed linearization of C4C10 YAC (data not shown). Analysis of HMW DNA, as above, indicated that no rearrangements had occurred (data not shown).
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Introduction of cYAC into LES cells
cYAC DNA was transfected into LES cells by lipofection. DNA preparations of both cYACs were isolated by PFGE and lipofected into LES1–5, LES2–4 and LES2–79 cells. After selection in G418, 45 colonies were picked for further growth (designated LC lines) from 3–6 x 106 cells transfected, giving an efficiency of 1.5 x 10–5 to 7.5 x 10–6. Because selection of colonies also required Cre-mediated lox recombination, it was difficult to distinguish recombination efficiency from transformation efficiency. Of the 45 colonies picked, 24 were harvested for genomic DNA and further analysis (Fig. 3).
Analysis of LC Lines
PCR analysis.
To determine the extent of cYAC integration in LC lines, genomic DNA from LC cells was amplified by PCR for a variety of loci. All 24 lines were PCR-positive for three loci: the recombined lox site, the HygR gene, and the yeast HIS3 gene. Gels of lox site PCR were Southern blotted and hybridized with the lox PCR product to confirm PCR results. LC lines were tested for AML1 exons 1, 7 and a, KCNE1 and several human chromosome 21 sequence tagged site (STS) markers. All PCR gels were Southern blotted and hybridized with either the purified PCR product or another appropriate probe (hIsk-I for KCNE1) to confirm PCR results. STS markers were tested but could not identify introduced human DNA due to cross-hybridization of primers to mouse sequences. Five of the twenty-four lines tested appear to contain full-length YACs: LC21, LC24, LC20, LC41 and LC43. Results of PCR on LC lines are summarized in Figure 3 and PCR primers are described in Table 1.
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Southern analysis.
HMW DNA was prepared from LC cells for analysis of cYAC integration and integrity. DNA was digested with NotI, NruI, EcoRI and BamHI. NotI and NruI digests were separated by PFGE. Blots were hybridized with probes hygromycin-B gene (Hyg), NcoI, Isk-I and AML1 exon 7. Analysis of BamHI digests reveals that LC lines have an altered restriction fragment length pattern compared with the original LES line when probed with Hyg, indicating integration of cYAC DNA at the lox site (Fig. 6A). Analysis of NotI digests (Fig. 6B) and NruI digests (data not shown) shows altered or additional bands in the LC lines when probed with Hyg, indicating the insertion of the YACs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A targeting system was designed to introduce circular YACs into defined sites within the ES genome by Cre-lox recombination. Two vectors containing single lox sites were designed in this study: (i) to introduce a single lox site into the ES genome; and (ii) to circularize linear YACs. Cre-lox recombination occurred between the two vectors to give the desired result of targeted integration of full-length YACs. Random integration of lox sites into the ES genome was chosen rather than targeted integration because the effect of chromatin structure on Cre-mediated recombination efficiency in vivo is unknown. Bronson et al. (11) chose the HPRT gene for integration of DNA into a lox site because it is a housekeeping gene and would therefore always be amenable to transcription. By targeting the site to a specific sequence, insertional mutagenesis of an essential gene by random integration will not occur; however, there is a risk that the inserted gene(s) may be influenced by the expression patterns of the target site. This is especially true if enhancers and promoters are known to affect nearby sequences.
This system is unique in many ways compared with previously used methods of introducing YACs into ES cells. A vector is described that allows for the stable circularization of linear YACs in yeast, and a method is described to isolate these circular molecules from total yeast DNA for lipofection into ES cells. Analyses of LC lines suggest that cYAC has fully integrated in five lines. Screening a wider panel of markers in the region may identify more human-specific primer sets and therefore allow for a more stringent analysis of YAC DNA present in LC lines.
The efficiency of YAC transfer in the creation and study of YAC transgenic mouse lines is often low compared with the effort required to make them. In lipofection of ES cells with YAC DNA, intact full-length YAC integrates randomly into the genome in ~10% of NeoR colonies generated (1). This efficiency is about the same as seen with microinjection (2–13%), which can involve a greater amount of DNA shearing, but is lower than that seen for spheroplast fusion (40%), which is complicated by the introduction of other yeast chromosomes into the mouse cell (2,5). The method presented here resulted in 21% of NeoR colonies generated having intact YAC sequence. This is a 2-fold increase in efficiency over similar lipofection methods. With improvements in cYAC isolation and lipofection techniques, even higher efficiencies will be possible. These techniques should greatly enhance our ability to produce and study transgenic mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Vector construction
pSF1Hyg.
A 1.4 kb XhoI fragment containing the Hyg gene under the control of the Polymerase II promoter was isolated from polIIHyB (courtesy of S. Fisher, Johns Hopkins University, Baltimore, MD). The 1.4 kb Hyg cassette was ligated into the single XhoI site in the vector pSF1 (12).
pLC2.
PCR primers containing SphI (pBRSph) and HindIII (pBRH3) sites were designed to amplify an 820 bp fragment from pBR322 (Table 1). The 820 bp PCR fragment was ligated into SphI and HindIII sites of pBS226 (12), creating p226pBR. A 1.2 kb BamHI fragment containing the yeast HIS3 gene was isolated from pHIS3pol2neo (courtesy of B. Lamb, Case Western Reserve University, Cleveland, OH) and ligated into the unique BamHI site of vector p226pBR.
YAC manipulation
General yeast protocols, media and standard genetic techniques were performed as described (13). To circularize linear YAC DNA, pLC2 was linearized with NruI and ScaI, leaving free ends homologous to linear YAC arms. YPH925 yeast containing linear C4C10 and C14B2 YAC DNA was transformed with linearized pLC2 by a standard lithium acetate transformation (1). Transformants were selected on SD8-HIS3 plates and SD8-URA3 plates.
Chromosome sized HMW yeast DNA was prepared as described (14) with modifications (1). Isolation of total genomic yeast DNA was as described (15). cYAC DNA molecules were subjected to electrophoresis, isolated as agarose plugs and digested with ß-agarase as described (1), with the following modifications. Conditions for electrophoresis were 48–60 h with the switch time ramped from 2 to 2.5 minutes. To isolate cYAC, the contents of the well and small band of 1% agarose below the well was cut out. One gram gel slices from this strip were melted at 70°C and very gently mixed such that the well contents were mixed with the agarose. Plugs were immediately re-solidified on ice and stored at 4°C. DNA plugs were digested with ß-agarase for lipofections as described (1). PFGE switch times were as follows: for karyotyping cC14B2 (16 h at 60 s then 14 h at 90 s); for karyotyping cC4C10 (14 h at 60 s then 10 h at 90 s); for preparative gels to isolate linear C4C10 (32 h at 30 s in 1% agarose); for preparative gels to isolate linear C14B2 (36 h at 60 s in 1.5% agarose) and for PFGE of digests of both YACs (8 h at 60 s then 15 h at 90 s).
Cell culture and blastocyst injections
The lipofection of plasmids into ES cells was performed essentially as described (16). For the transfection of pSF1Hyg into ES cells, 2 µg of pSF1Hyg was co-lipofected with 4 µg of pBS185. Control transfections of pLC2 into LES cells were performed as follows. For some transfections, 2–3 µg of pLC2 was co-lipofected with 4–10 µg of pBS185; for others, 20 µg of pLC2 and 10 µg of pBS185 were co-electroporated into 1 x 107 LES cells (in 700 µl of ES medium) at 450 V/500 µF. Lipofection of cYAC DNA into ES cells was performed using the suspension method (16). ES cells were selected in either 250 µg/ml G418 (Geneticin; Life Technologies, Rockville, MD), or 200 µg/ml hygromycin-B (Boehringer Mannheim, Indianapolis, IN). Genomic DNA was isolated from ES cells and mouse tails by a standard salting out procedure (17), and HMW DNA was isolated from ES cells in agarose plugs as described (18). Injection of dissociated ES cells into C57BL/6J blastocysts and matings of resultant chimeras were carried out as described (19).
DNA analysis
PCR primer sequences and cycling conditions for STS markers are located on the Eleanor Roosevelt Institute Chromosome 21 STS Database web page (http://www-eri.uchsc.edu/chr21/erists.html ). The following primer sets are described in the associated reference: AML1 exon a (20), AML1 distal promoter (21) and DSCR1 (22). Other PCR primers and conditions are listed in Table 1. The primer LoxPCR-1 (5'-TGGAGACGCCATCCACGCTG-3') was used to sequence the PCR product of loxF and loxR primers. This is a fusion product from the vectors pSF1Hyg and pLC2.
For standard Southern blot analysis (23), DNA was digested with the appropriate restriction enzyme(s) and subjected to electrophoresis in 1% agarose overnight. Probes were labeled by the random hexamer labeling method (24) using the random primed DNA labeling kit (Boehringer Mannheim) and hybridized at 65°C (25). Oligonucleotides were end-labeled using T4 polynucleotide kinase with forward reaction buffer (Life Technologies).
FISH and karyotyping
Metaphase spreads were prepared from colchicine-treated mouse ES cells by standard methods (26). Standard G-banding methods were used to analyze the modal chromosome number of ES lines prior to microinjection. For FISH, slides were pretreated in 2x SSC at 37°C for 30 min, dehydrated and denatured for 5 min at 75°C before hybridization at 37°C overnight. Plasmid DNA (pSF1Hyg and pLC2) and total yeast genomic DNA were used as probes. Probes were labeled and two-color FISH analysis was performed as described (27).
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ACKNOWLEDGEMENTS |
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The authors would like to thank Bruce Lamb for his experience in the field of YAC transgenesis, which made this work possible; Ann Lawler for critical reading of the manuscript; and Brian Sauer, Phil Heiter and Forrest Spencer for plasmids, yeast strains and advice. This work was supported by grant HD24605 (J.D.G., C.S.M., G.S.). L.M.C. was a student in the predoctoral training program in human genetics and supported by the grant GM07814.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Departments of Obstetrics and Gynecology, and Pathology, Thorn 625, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA. Tel: +1 617 732 7980; Fax: +1 617 738 6996; Email: lcall@rics.bwh.harvard.edu
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