Biotech 1998
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Benchmarks PCR-Based Strategy for Genotyping Mice and ES Cells Harboring LoxP Sites BioTechniques 25:968-972 (December 1998) Increasingly, the Cre-loxP system is being used to generate both null and conditional mutations in mice by ho- mologous recombination in embryonic stem (ES) cells (1,3–5). The advantage of the Cre-loxP approach in generating mutations in mice, is that the gene of interest can be inactivated in any organ at a given time during the mouse’s life, thus bypassing developmental and/or pleiotropic effects inherent in the clas- sical knock-out approach. Furthermore, this strategy allows for the generation of null alleles as well as “floxed” alle- les (genomic DNA flanked by loxP sites) by using just one construct for gene targeting, since one parental ho- mologous recombinant ES clone can yield subclones with both null and floxed alleles, following transient trans- fection with a Cre-recombinase ex- pressing plasmid. Genotype analysis of mutant mice, generated by homologous recombination is done using genomic DNA for either Southern analysis or by use of the polymerase chain reaction (PCR). PCR is preferred since it rapidly yields the required results and allows for a larger throughput of samples. For classical knock-out techniques, where a large marker gene such as a neomycin- resistance gene or the lacZ gene (or both) is either inserted into or replaces an exon(s) (2), PCR analysis requires the use of three primers (see below). In our experience, amplifying mouse genomic DNA is not straightforward, because determining the correct condi- tions with respect to annealing temper- ature, magnesium concentration, quali- ty and amount of template, are all critical factors in allowing for the de- velopment of a robust assay. The more primers used for this type of assay, the more difficult the task of determining the optimal PCR conditions. We have been using the Cre-loxP system to gen- erate both null and conditional muta- tions in a number of genes, including the following transcription factors: cAMP responsive element binding pro- tein (CREB), glucocorticoid receptor (GR) and hepatocyte nuclear factor 4γ (HNF4γ ). Here, we describe the PCR strategy we use in analyzing the geno- types of our floxed mice, using only two primers, making the development of assays simpler, quicker and more ro- bust. During our analysis of CREB and HNF4γ ES cell clones, we realized that this approach also allowed rapid screening for multicopy insertions of the targeting construct in putative ho- mologous recombined ES clones. For the classical knock-out ap- proach, analysis of mouse genotypes by PCR requires use of three primers, one common to both mutant and wild-type (WT) alleles and one specific to each (Figure 1A; primers 1, 2wt and 3mut) allowing for the amplification of rela- tively short products in each case. The amplificati on yields two different sized products, one mutant specific (null) and one WT specific. Use of only two primers on either side of the selection cassette in this case is problematic, be- cause the size of the reporter gene(s) is relatively large (>1 kb), making routine amplification of large genomic frag- ments in multiple heterogeneous sam- ples difficult. Secondly, the large size difference, hence amplification rate dif- ference, between the mutant and WT products in this case would further complicate the determination of suit- able PCR conditions. For floxed mice, genotyping by PCR is possible using only two primers flanking one loxP site (Figure 1B; primers 1 and 2). This allows for the amplification of two different sized products depending on the presence or absence of the loxP site. The size dif- ference between the floxed allele and WT allele is due both to the extra nu- cleotides that comprise the loxP se- quence (34 bp) and the surrounding re- striction sites used for constructing the targeting construct. Because the size difference is relatively small, differen- tial amplification of one product over the other is avoided. Moreover, by de- signing primers outside of both loxP 968 BioTechniques Vol. 25, No. 6 (1998) Figure 1. Strategies for genotype analysis by PCR. (A) Both WT and null alleles are shown, as generated by the classical knock-out strategy; in this case, the gene is silenced by the in-frame in- sertion-replacement into the indicated exon by a neomycin-resistance and lacZ reporter gene. Po- sitions of primers used for the amplification and detection of differences between the two alleles are shown. Primer 1 is common to both loci, the 2wt primer is WT-specific, and the 3mut primer is mutant-specific. (B) Both WT and correspond- ing floxed alleles are shown. The loxP sites are shown as gray rectangles, whereas primers are shown as arrows and exons as black rectangles. See text for further explanation. Figure 2. Amplification of DNA. Amplified products from PCR assays containing genomic DNA from CREB-loxP mice (lanes 1–3), ES cells (lanes 4–6) and HNF4γ -loxP ES cells (lanes 7–9) as template. A 10-µL sample of each PCR was electrophoresed on a 1.2% agarose gel containing 0.1 µg/mL EtdBr. Lane 1, homozygous (floxed); lanes 2, 5 and 8, heterozygous; lanes 3, 4 and 7, WT; lanes 6 and 9, het- erozygous with multiple copies of loxP targeting vector. PCR primers used: CREB-loxP; sense, 5′- TATGTAAAGCAAGGGAAGATAATG-3′; antisense, 5′-TAGACATACTTGACCCATAGCATT-3 ′; HNF4γ -loxP; sense, 5′-TTGAGTGTTCTTCACATTAACACC-3 ′; antisense, 5′- CTGCATCCATAC AA- CAACTATTTT-3 ′.
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Benchmarks
PCR-Based Strategy for
Genotyping Mice and ES
Cells Harboring LoxP
Sites
BioTechniques 25:968-972 (December 1998)
Increasingly, the Cre-loxP system isbeing used to generate both null andconditional mutations in mice by ho-mologous recombination in embryonicstem (ES) cells (1,3–5). The advantageof the Cre-loxP approach in generatingmutations in mice, is that the gene of interest can be inactivated in any organat a given time during the mouse’s life,
thus bypassing developmental and/orpleiotropic effects inherent in the clas-sical knock-out approach. Furthermore,this strategy allows for the generationof null alleles as well as “floxed” alle-les (genomic DNA flanked by loxPsites) by using just one construct forgene targeting, since one parental ho-mologous recombinant ES clone canyield subclones with both null andfloxed alleles, following transient trans-fection with a Cre-recombinase ex-pressing plasmid. Genotype analysis of mutant mice, generated by homologous
recombination is done using genomic
DNA for either Southern analysis or byuse of the polymerase chain reaction(PCR). PCR is preferred since it rapidlyyields the required results and allows
for a larger throughput of samples. Forclassical knock-out techniques, where alarge marker gene such as a neomycin-resistance gene or the lacZ gene (orboth) is either inserted into or replacesan exon(s) (2), PCR analysis requiresthe use of three primers (see below). Inour experience, amplifying mousegenomic DNA is not straightforward,because determining the correct condi-tions with respect to annealing temper-ature, magnesium concentration, quali-ty and amount of template, are allcritical factors in allowing for the de-
velopment of a robust assay. The moreprimers used for this type of assay, themore difficult the task of determiningthe optimal PCR conditions. We havebeen using the Cre-loxP system to gen-erate both null and conditional muta-tions in a number of genes, includingthe following transcription factors:cAMP responsive element binding pro-tein (CREB), glucocorticoid receptor(GR) and hepatocyte nuclear factor 4γ (HNF4γ ). Here, we describe the PCRstrategy we use in analyzing the geno-types of our floxed mice, using only
two primers, making the developmentof assays simpler, quicker and more ro-bust. During our analysis of CREB andHNF4γ ES cell clones, we realized thatthis approach also allowed rapidscreening for multicopy insertions of the targeting construct in putative ho-mologous recombined ES clones.
For the classical knock-out ap-proach, analysis of mouse genotypes byPCR requires use of three primers, onecommon to both mutant and wild-type
(WT) alleles and one specific to each(Figure 1A; primers 1, 2wt and 3mut)allowing for the amplification of rela-tively short products in each case. Theamplification yields two different sizedproducts, one mutant specific (null) andone WT specific. Use of only twoprimers on either side of the selectioncassette in this case is problematic, be-cause the size of the reporter gene(s) isrelatively large (>1 kb), making routineamplification of large genomic frag-ments in multiple heterogeneous sam-ples difficult. Secondly, the large size
difference, hence amplification rate dif-ference, between the mutant and WTproducts in this case would furthercomplicate the determination of suit-able PCR conditions.
For floxed mice, genotyping byPCR is possible using only two primersflanking one loxP site (Figure 1B;primers 1 and 2). This allows for theamplification of two different sizedproducts depending on the presence orabsence of the loxP site. The size dif-ference between the floxed allele andWT allele is due both to the extra nu-
cleotides that comprise the loxP se-quence (34 bp) and the surrounding re-striction sites used for constructing thetargeting construct. Because the sizedifference is relatively small, differen-tial amplification of one product overthe other is avoided. Moreover, by de-signing primers outside of both loxP
968 BioTechniques Vol. 25, No. 6 (1998)
Figure 1. Strategies for genotype analysis by
PCR. (A) Both WT and null alleles are shown, asgenerated by the classical knock-out strategy; in
this case, the gene is silenced by the in-frame in-sertion-replacement into the indicated exon by a
neomycin-resistance and lacZ reporter gene. Po-sitions of primers used for the amplification and
detection of differences between the two allelesare shown. Primer 1 is common to both loci, the
2wt primer is WT-specific, and the 3mut primeris mutant-specific. (B) Both WT and correspond-
ing floxed alleles are shown. The loxP sites areshown as gray rectangles, whereas primers are
shown as arrows and exons as black rectangles.See text for further explanation.
Figure 2. Amplification of DNA. Amplified products from PCR assays containing genomic DNA from
CREB-loxP mice (lanes 1–3), ES cells (lanes 4–6) and HNF4γ -loxP ES cells (lanes 7–9) as template. A10-µL sample of each PCR was electrophoresed on a 1.2% agarose gel containing 0.1 µg/mL EtdBr.
Lane 1, homozygous (floxed); lanes 2, 5 and 8, heterozygous; lanes 3, 4 and 7, WT; lanes 6 and 9, het-erozygous with multiple copies of loxP targeting vector. PCR primers used: CREB-loxP; sense, 5′-TATGTAAAGCAAGGGAAGATAATG-3′; antisense, 5′-TAGACATACTTGACCCATAGCATT-3′;HNF4γ -loxP; sense, 5′-TTGAGTGTTCTTCACATTAACACC-3 ′; antisense, 5′- CTGCATCCATACAA-
CAACTATTTT-3′.
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Benchmarks
sites, one can use the assay for deter-mining recombination mediated by Crerecombinase (not shown). Results of PCR assays are shown for genomic
DNA from mouse tails and ES cells forthe CREB-loxP allele, and from EScells for the HNF4γ -loxP allele. TheDNAs from both ES cells (approxi-mately 106 cells) and mouse tails (5-mm biopsy) were prepared using thesame method. We chose to use amethod that allows for the productionof relatively large amounts of DNA,since we analyze the samples for othertransgenes using other techniques. Al-ternative methods, requiring much lessstarting material and less handlingcould be used. The DNA preparations
were made and analyzed as follows: (i)Cells or tail material were suspended in0.8 mL lysis buffer (1% sodium dode-cyl sulfate [SDS], 50 mM Tris-HCl, pH8.0, 100 mM EDTA, 200 µg/mL pro-teinase K) and incubated at 56°C for 16
h. (ii) A 0.25-mL vol of saturated NaClwas added, and the samples weremixed on a Thermomixer (Eppendorf,Hamburg, Germany) for 5 min. (iii) Af-
ter centrifugation for 10 min (15000×g), 0.8 mL of the supernatant was pre-cipitated with 0.6 mL isopropanol. (iv)The DNA was resuspended in 0.1 mLTE (50 mM Tris-HCl, pH 8.0, 10 mMEDTA). (v) For amplification, 1 µLDNA, (1:100 dilution) was added to amixture containing PCR buffer (Boeh-ringer Mannheim GmbH, Mannheim,Germany), 2.5 mM magnesium chlo-ride, 0.2 mM of each dNTP (Boeh-ringer Mannheim); 25 pmol of eachprimer and 0.5 U Taq DNA Polymerase(Boehringer Mannheim). Amplification
was performed in a Gradient 96 Robo-Cycler® (Stratagene, La Jolla, CA,USA) using the following conditions:95°C for 5 min, followed by 35 cyclesof 95°C for 40 s, 60°C for 1 min and72°C for 40 s. (vi) Ten microliters of
the sample were then electrophoresedon a 1.2% agarose gel (Life Technolo-gies, Paisley, Scotland, UK) containing0.1 mg/mL ethidium bromide (EtdBr).
For CREB-loxP, the amplified productof the floxed allele was 568-bp long;while for the WT allele, the band seenmigrated at 500 bp. For HNF4γ -loxP,the corresponding bands were 512 and400 bp, respectively (Figure 2). Thelarger size of the loxP products com-pared with WT was due to the loxPsites (34 bp) and sequences surround-ing the loxP sites, which include re-striction sites coming from the parentalplasmids. The assay has proven to berobust and consistent, given that we canroutinely screen one hundred or more
samples in one assay.A further advantage of this PCR ap-
proach is the rapid identification of un-desirable multicopy insertions of theloxP targeting vector when incorporatedinto putative homologously recombined
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Benchmarks
ES cells. This phenomenon has beenobserved in our laboratory in a numberof cases. Clones putatively identified ashomologous recombinants by Southern
analysis can be quickly checked for ran-dom insertion of extra copies of the tar-geting vector by PCR. Given the smalldifference in size between the two alle-les and use of only two primers com-mon to both WT and mutant DNA, theamplification rate of both products issimilar. Therefore, any significant dif-ference in signal intensity between theWT and floxed products indicates mul-tiple integration of the targeting vector,which can be later confirmed by South-ern analysis. The presence of multicopyinsertions in CREB-loxP ES cells and
HNF4γ -loxP ES clones resulted in over-amplification of the mutant band (Fig-ure 2, lanes 6 and 9). Presence of multi-ple copies of the mutant constructs wasconfirmed by Southern blot analysis(data not shown).
To summarize, with the ever increas-ing use of the Cre-loxP system studyinggenetic mutations in the mouse, we pre-sent a generally applicable PCR-basedapproach for genotyping genomic DNAharboring loxP sites. Unlike previouslyreported knock-out detection methods,where PCR analysis for genotyping re-
quires use of three primers, only twoprimers are required here, allowing forrobust PCR assays to be set up rapidly.Due to the use of identical primers inthe detection of both WT and loxP alle-les, the method also allows for accuratedetection of copy number differenceswhen screening ES cells.
REFERENCES
1.Betz, U.A., C.A. Vosshenrich, K. Rajewskyand W. Muller. 1996. Bypass of lethality with
mosaic mice generated by Cre-loxP-mediated
recombination. Curr. Biol. 6 :1307-1316.2.Bronson, S.K. and O. Smithies. 1994. Alter-
ing mice by homologous recombination usingembryonic stem cells. J. Biol. Chem.
269:27155-27158.
3.Chambers, C.A. 1994. TKO’ed: lox, stock and barrel. Bioessays 16 :865-868.
4.Terry, R.W., L. Kwee, H.S. Baldwin andM.A. Labow. 1997. Cre-mediated generationof a VCAM-1 null allele in transgenic mice.
Transgenic Res. 6 :349-356.5.Tsien, J.Z., D.F. Chen, D. Gerber, C. Tom,
E.H. Mercer, D.J. Anderson, M. Mayford,
E.R. Kandel and S. Tonegawa. 1996. Subre-gion- and cell type-restricted gene knockout in
mouse brain. Cell 87 :1317-1326.
We thank Werner Fleischer for oligonu-
cleotide synthesis and Dr. A. Paula Mon-
aghan for critically reading the manuscript.
Address correspondence to Prof. Günther
Schütz, Division Molecular Biology of theCell I, German Cancer Research Centre,
Im Neunheimer Feld 280, 69120 Heidel-
berg, Germany. Internet: g.shuetz@dkfz-
heidelberg.de
Received 9 June 1998; accepted 17 Au-
gust 1998.
Theo Mantamadiotis, StavrosTaraviras, François Troncheand Günther SchützGerman Cancer Research
Centre Heidelberg, Germany
Adaptation of the
Fluorogenic 5′′-Nuclease
Chemistry to a PCR-
Based Reverse
Transcriptase Assay
BioTechniques 25:972-975 (December 1998)
Assays for the enzymatic activity of reverse transcriptase (RT) are used asgeneral methods for the detection of both known and unknown retroviruses.These conventional RT assays measurethe direct incorporation of a labeled de-oxynucleotide into cDNA on a ho-mopolyribonucleotide template. How-
ever, these RT assays suffer from a lack of sensitivity when compared with se-quence-specific assays such as RNA-polymerase chain reaction (PCR). Thesensitivity of the RT assay was in-creased by using an RNA template of known sequence and PCR to amplifythe cDNA product. These PCR-basedRT (PBRT) assays (3,14,15) are at leasta million fold more sensitive than con-ventional RT assays and have the ca-pacity to detect a single retrovirus par-ticle. These assays have also beencalled PERT, for product-enhanced RT,
(14) and Amp-RT (3).Although these PBRT assays have
the potential to be useful in many fieldsof retrovirology, several features of theassays have limited their general use.Because some thermostable DNA poly-merases have RNA-dependent DNApolymerase activity, background sig-nals can be generated (10,14). Addi-tionally, the assay is not selective forretroviruses, because lysates of all cellstested have PBRT activity (9,10,15). Fi-nally, the assay is labor-intensive, in-convenient to perform and quantitativeover only a limited range.
Because the cDNA detection step inthe PBRT assay is time-consuming, inthis paper we describe the adaptation of this part of the PBRT assay to the 5′-nu-clease (TaqMan®) technology for use inthe PRISM® 7700 Sequence DetectionSystem (PE Applied Biosystems, Fos-ter City, CA, USA; References 2 and 8).The modified assay, which we call theTM-PERT assay, not only expedites thedetection of the PCR product, but alsoprovides a linear reaction over at least
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