Tech Notes
Faster Genotyping of Transgenic Mice with the MasterPure™ DNA
Purification Kit & MasterAmp™ PCR Products
Judith T. Schanke, Epicentre Technologies
Introduction
Introduction Transgenic mice carry a
segment of foreign DNA that has been
incorporated into their genomes by
either homologous recombination or by a
nonhomologous insertion method, such
as pronuclear microinjection. The incorporated transgene is detected by genomic
DNA isolation from a tissue sample, followed by PCR amplification of a region of
the inserted gene. The genomic DNA tissue
source most commonly used for genotyping is mouse tail, however, ear, toe, hair,
and blood samples are also used.1,2
Typically, genomic DNA isolation procedures from tail slices involve long incubations (one hour to overnight) at high temperature in the presence of proteinase K, or
require extensive homogenization of the
sample prior to cell lysis. Here, we describe
a fast and reliable method of obtaining
high molecular weight genomic DNA from
a series of mouse tail tissue samples. The
MasterPure™ DNA Purification Kit enables
genomic DNA purification without lengthy
incubations, hazardous organic solvents,
or cumbersome spin columns. We show
that the high DNA yields obtained with
the MasterPure DNA Purification Kit, in
combination with the MasterAmp™ PCR
Optimization Kit, enables fast, efficient
genotyping of transgenic mice.
Elaborate systems have been developed to
study the effects of specific transgene expression. Bacterial recombinases are commonly used to drive site-specific recombination in mammalian cells, allowing for
conditional gene inactivation in a specific
subset of cells.3 One such system is the
Cre/lox P recombination system.4 Cross
breeding transgenic mice containing the
Cre recombinase under the control of a
tissue specific promoter and a transgene
flanked by lox P sites (a “floxed” gene)
allows the tissue specific knockout of a
gene of interest. In this study, we demonstrate genotyping of transgenic mice
bred to carry the Cre recombinase gene
and a “floxed” transgene. We also demonstrate that mouse tail DNA purified using
the MasterPure Kit is suitable for use in
genotyping of transgenic mice using
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the SV40 T Ag system. The expression of
specific sequences from the small DNA
virus, SV40, deregulates cell growth and
immortalizes cells. When linked to a tissuespecific promoter or enhancer element, cell
immortalization and subsequent neoplastic transformation can be directed to
specific cell types. Here, we genotype mice
cross bred to contain the SV40 T Ag and a
“floxed” transgene.
Methods
Isolation of genomic DNA from mouse
tail tissue using the MasterPure DNA
Purification Kit
Mouse tail tissue samples screened for the
Cre recombinase, SV40 T Ag, and “floxed”
transgenes were kindly provided by Dr.
Wilhelmine N. de Vries and Dr. Barbara
Knowles, The Jackson Laboratory, Bar
Harbor, ME.
Frozen mouse tail tissue samples, approximately 1-2 mm in length, were placed in
1.5 ml tubes and suspended in 300 µl of
Tissue and Cell Lysis Buffer containing 50
µg of proteinase K. The samples were incubated at 65°C for 15 minutes, with vortex
mixing every 5 minutes.
After lysis, the standard protocol for the
MasterPure DNA Purification Kit was followed.5 Briefly, the samples were placed on
ice for 3 minutes and then 150 µl of Protein
Precipitation Reagent were added. The
samples were vortex mixed, the proteins
were pelleted by full-speed microcentrifugation for 10 minutes, and the nucleic
acid-containing supernatant was transferred to a new tube. Total nucleic acid was
then precipitated by the addition of 500 µl
of isopropanol, samples were inverted 30
times, and pelleted by microcentrifugation
at full-speed for 10 minutes. The isopropanol was decanted and the nucleic acid
pellet was gently washed twice with 70%
ethanol. All residual ethanol was removed
by aspiration and the pellet was resuspended in 35 µl of TE buffer.
Optimization of transgene
coamplifications
The sequences used to amplify the 490 bp
region of the SV40 T Ag transgene were:
SV1, 5′-GGACAAACCACAACTAGAATGCAGTG-3′ and SV5, 5′-CAGAGCAGAATTGTGGAGTGG-3′. The sequences of the Cre
recombinase primers were: 5′-TGATGAGGTTCGCAAGAACC-3′ and 5′-CCATGAGTGAACGAACCTGG-3′. The sequences of the
primers that detect the “floxed” transgene
were: 5′-AAGGTAGAGTGATGAAAGTTGTT-3′
and 5′-CACCATGTCCTCTGTCTATCC-3′.
In order to determine the best amplification conditions for each primer pair,
12 reactions were assembled with the
Figure 1. Optimization of the PCR coamplification of SV40 T Ag and a “floxed” transgene from mouse
genomic DNA with the MasterAmp PCR Optimization Kit. Lanes A-L show the amplification products
resulting from multiplex PCR using the 12 MasterAmp PCR Optimization Kit PreMixes. M= 100 bp DNA
ladder.
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MasterAmp PCR Optimization Kit PreMixes,
which vary in their concentrations of
MgCl2 and MasterAmp PCR Enhancer (with
betaine)*. The 50 µl reactions contained
50-100 ng of genomic DNA, 12.5 pmoles of
each primer, 1.25 U of MasterAmp Taq DNA
Polymerase, and one of the MasterAmp
PCR PreMixes A-L, (containing 1X PCR buffer, 200 µM each dNTP, 1.5-3.5 mM MgCl2,
and 0-4X MasterAmp PCR Enhancer).
The cycling profile used for the coamplification of the SV40 T Ag and the “floxed” transgene was 2 minutes at 94°C, then 40 cycles
of 94°C for 1 minute, 61°C for 1 minute, and
72°C for 1 minute, followed by a 4-minute
extension at 72°C. Fifteen percent of the
total multiplex reaction volume (7.5 µl) was
separated on a 2% agarose gel, stained
with ethidium bromide and visualized by
transillumination. The optimization of the
coamplification of the SV40 T Ag and the
“floxed” transgene is depicted in Figure 1.
The reaction with MasterAmp PCR PreMix E
(Figure 1, Lane E), containing 2.5 mM MgCl2
and 2X MasterAmp PCR Enhancer, (as well
as 1X PCR buffer and 200 µM each dNTP),
was chosen for genotyping all samples with
these primer pairs.
A similar multiplex PCR optimization analysis was performed on DNA isolated from
a litter of mice bred to contain both the
Cre recombinase and the “floxed” transgenes. The amplification reactions, cycling
conditions, and electrophoresis conditions
were the same as those described above.
MasterAmp PCR PreMix B (containing
1.5 mM MgCl2 and 0X MasterAmp PCR
Enhancer) was chosen for the multiplex
genotyping of the Cre recombinase gene
and the “floxed” transgenes.
Results
Genomic DNA purification
The 1-2 mm samples (1-5 mg) of tail tissue
yielded 500 ng - 2 µg of genomic DNA in
15 minutes without homogenization or
waiting for the full dissolution of tail slices.
DNA was quantitated by fluorimetry with
the Hoechst H 33258 dye, which binds
double-stranded DNA. The yields increased
to more than 10 µg when the incubation
time in the presence of proteinase K was
increased to 60 minutes, with vortex mixing
every five minutes. Even higher yields can
be obtained with homogenization of the
sample, however, these procedures are unnecessary unless genotyping by Southern
blot is required. A 30-minute incubation at
37°C with 5 µg of RNase A (included in the
MasterPure DNA Purification Kit) can also
be included to remove cellular RNA, but is
unnecessary for most standard or multiplex
PCR genotyping.
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Figure 2. Coamplification products of SV40
T Ag and a “floxed” transgene from genomic
DNA samples. The genotypes of 6 transgenic
mice. M, 100 bp DNA ladder.
Figure 3. Coamplification products of Cre
recombinase and a “floxed” transgene from
genomic DNA samples. The genotypes of 6
transgenic mice. M, 100 bp DNA ladder.
Genotyping mouse tail DNA for the
presence of SV40 T Ag and “floxed”
transgenes
The amplification of a region of the SV40 T
Ag with the SV1 and SV5 primer pair results
in a product of approximately 490 bp.
Mice not containing the “floxed” transgene
produce a 221 bp band upon amplification, denoted as a +/+ genotype. If the
“floxed” transgene is inserted (flx/flx), a 324
bp product will result. The flx/+ genotype
results in a multiplex pattern with both the
221 bp and the 324 bp products. Most of
the possible combinations of transgenic
progeny were detected in the litters examined (Figure 2). Samples 4 and 5 contain
both T Ag and the “floxed” transgenes
(SV40/+, flx/+).
15-minute incubation to lyse and proteinase K-treat the sample, produces ample
DNA for several (5-20) PCR analyses. This
rapid protocol can also be used to isolate
sufficient DNA from other genotyping
tissue sources, such as toes, hair, and ears.
Simple protocol modifications allow for the
isolation of enough intact genomic DNA for
Southern analysis, if desired.
The optimization of PCR amplification of
transgenes with new primers is fast and
convenient with the use of the MasterAmp
PCR Optimization Kit and MasterAmp
Taq DNA Polymerase. The combination
produces abundant amplification products,
even in the presence of RNA or with multiple primer pairs present in the reaction.
Therefore, the MasterPure DNA Purification
Kit and the MasterAmp PCR products allow
quick, convenient, and reliable genotyping
results.
Genotyping mouse tail DNA for the
presence of Cre recombinase transgene
and a “floxed” transgene.
The presence of the Cre recombinase gene
is detected by the amplification of a 400 bp
product. The “floxed” products expected
(221 bp and 324 bp) were as described
above. The genotyping results are depicted
in Figure 3. Samples 4 and 5 clearly contain
both the Cre recombinase and the “floxed”
transgenes (Cre/+, flx/flx).
Summary
The MasterPure DNA Purification Kit is the
quickest, most reliable method available
to isolate genomic DNA for transgenic
genotyping. The protocol, requiring only a
References
1. Malumbres, M. et al. (1997) Biotechniques 22 (6), 1114.
2. Abbott, C. (1993) in: Transgenesis Techniques. Principles and Protocols. Humana
Press. D. Murphy and D.A. Carter (Eds.),
317.
3. van der Neut, R. (1997) J. Neurosci.
Methods 71, 19.
4. Pluck, A. (1996) Int. J. Exp. Pathol. 77,
269.
5. Schanke, J.T. and Watson, J. (1998)
Epicentre Forum 5 (2),12.
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