Transgenics, Vol. 4, pp. 121-135
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© 2004 Old City Publishing, Inc.
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A Novel Gene Transmission Pattern of Exogenous
DNA in Offspring Obtained After Testis-Mediated
Gene Transfer (TMGT)
M A S A H I R O S AT O a * , S H I N G O N A K A M U R A b , c
a
The Institute of Medical Sciences, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan
b
Division of Nephrology and Metabolism, School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan
c
Present address: Department of Surgery II, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan
We have attempted to establish a new method,
so-called "testis-mediated gene transfer,
TMGT", based upon gene transfer via direct
introduction of exogenous plasmid DNA into
a testis as an alternative to microinjectionmediated transgenesis. We found that i) high
transmission rate of exogenous DNA to
offspring is achieved after TMGT, ii) the
pattern of gene transmission from F0 to F2
generations is non-Mendelian, iii) the copy
number of exogenous DNA is below 1 copy
per diploid cell, and iv) gene expression does
not occur or is very slight if it occurs. In this
study, we examined the pattern of gene
transmission of exogenous DNA in the
offspring (at F0 to F2 generations) obtained
after TMGT. A complex (70 µl) of circular
pCAG/NCre plasmid (a Cre expression
plasmid; 8 µg/testis) and FuGENE™6 (16
µ l/testis; Boehringer Mannheim GmbH,
Germany) was injected into the testes of
transgenic mice carrying a loxP-flanked
enhanced green fluorescent protein cDNA
sequence (termed CETZ-17) 3 times 3 days
apart. On 7 to 21 days after final injection,
these injected males were mated to
superovulated CETZ-17 females to obtain F0
pups. From these mice, F1 and F2 offspring
were obtained. Genotyping of these mice was
performed by PCR using several primers
recognizing several parts of pCAG/NCre. We
observed the following: i) at least two types of
pCAG/NCre which included intact plasmid
and deleted form (lacking pBluescript SK(-)
____________________
*Corresponding author: [email protected]
121
122
vector backbone) of plasmid in F0 offspring,
ii) during transition of F0 to F1, conversion of
the intact form to the deleted one occurred
frequently, and iii) the deletion appeared to
occur outside the 1.3-kb NCre gene (probably
at a portion containing CAG promoter and
the 1st intron of the chicken β-actin gene, and
a portion containing the 3’-noncoding region
of the rabbit β-globin gene and SV40 poly(A)
signals). These findings suggest i) the possible
occurrence of autosomal proliferation of
plasmid or its survival in mouse tissues during
TMGT-mediated gene delivery, and ii) the
presence of a regulated mechanism to elicit
deletion of the exogenous plasmid
pCAG/NCre probably at defined sites.
Key Words: Episome, Gene transmission, In
vivo gene transfer; PCR, Sperm vector, Testis
INTRODUCTION
Lavitrano et al. [1] reported that mouse
spermatozoa incubated with DNA-containing
medium could serve as vectors for introducing
exogenous DNA into ova. This technology is
therefore termed “sperm-mediated gene transfer
(SMGT)”, and is a unique system for efficient
production of transgenic animals because of its
simplicity. However, contradictory results
regarding successful production of transgenics by
SMGT have been reported by other investigators
working in several mammalian species [2-7].
Recently, Lavitrano and her collaborators
demonstrated that SMGT is repeatable [8].
We have examined another possible method of
gene transfer in vivo, so-called “testis-mediated
gene transfer (TMGT)” by introduction of foreign
DNA into testes [9-13]. With this approach,
exogenous DNA introduced directly into testis
appeared to be mainly uptaken by testicular
TRANSGENICS
S AT O A N D N A K A M U R A
spermatozoa, which subsequently transferred the
DNA to oocytes through fertilization. Initial
experiments were conducted by direct injection of
Ca-phosphate-precipitated plasmid DNA, but this
attempt failed to produce transgenic mice [9].
However, we later showed that a single injection of
circular plasmid DNA complexed with
Lipofectin™ into mature mouse testes is sufficient
for transfection of spermatozoa (epididymal
spermatozoa), and for relatively high efficiency of
gene delivery to mid-gestational fetuses (F0)
obtained by mating of injected males with normal
females [11]. We also found in that study that i) the
DNA introduced may have been present mosaically
in fetal tissues, since it was estimated to be present
at less than 1 copy per diploid cell [11], and ii) the
DNA introduced was transmitted at least to the
second generation [12]. Expression of the DNA
introduced was first evident only in F0 early
blastocysts, but almost absent in F0 midgestational fetuses and organs of adult F0 mice
[10,12]. We failed to detect any gene expression in
these F0 fetuses at Northern blot level, and
succeeded in detecting it only in a limited number
of samples when nested RT-PCR (a very sensitive
method for detection of mRNA) was performed
[12]. Furthermore, we tested several commercially
available reagents used for in vitro gene transfer to
examine which is best for introducing high
numbers of copies of exogenous DNA into the fetal
mouse genome via TMGT [13]. Unfortunately, we
found no candidate reagents for this purpose.
We recently assessed the mechanisms of TMGT
in greater detail by injection of trypan blue (TB), a
dye generally used for staining dead cells in cell
culture systems, and Hoechst 33342, a fluorescent
dye generally used in staining cell nuclei, into adult
murine testes, and found that the solution
introduced into testis is transported to the ducts of
the caput epididymis via the rete testis and efferent
ducts immediately after testis injection and reaches
the corpus and cauda epididymis within 3-4 days
T E S T I S - M E D I AT E D G E N E T R A N S F E R ( T M G T )
after injection [14]. These findings suggest that
exogenous DNA introduced directly into testis was
mainly uptaken by epididymal spermatozoa, which
subsequently transferred the DNA to oocytes
through fertilization.
We previously noted that the exogenous gene
(circular plasmid) introduced into a testis was in
fact transmitted to F1 and F2 generations, but
that the pattern of gene transmission from one
generation to the next was non-Mendelian [12].
In this study, we examined this phenomenon in
detail and found that the introduced plasmid
DNA exsisted as an intact form or deleted form
lacking a large part of plasmid sequence including
vector backbone in the offspring obtained by
TMGT. Interestingly, the deletion appeared not to
occur randomly, but instead to be confined to
certain specific sites on a plasmid, suggesting the
presence of unknown, but precisely regulated
mechanisms to cleave the exogenous plasmid in
the TMGT system.
MATERIALS AND METHODS
Testis Injection of Plasmid DNA Mixed
With Lipid
The circular Cre expression vector
pCAG/NCre (Figure 1; [15]) was used for testis
injection. pCAG/NCre consists of the
cytomegalovirus enhancer and chicken β-actin
promoter (termed “CAG”; [16]), 1st intron of
chicken β-actin promoter, the bacteriophage P1
Cre gene [with the sequence for nuclear location
signal (NLS) attached to its 5’ end; hereafter
referred to as NCre], a portion of the 2nd intron,
3rd exon and 3’-noncoding region of the rabbit βglobin gene, SV40 poly(A) signals, a portion of
pBR322, and pBluescript SK(-) (Stratagene, La
Jola, CA). Injection of the DNA-containing
solution into testes of CETZ-17 transgenic mice
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(with B6C3F1 genetic background) aged 3-6
months was performed as described previously
[12]. CETZ-17 transgenic mice carry 2-3 copies
of transgenes consisting of CAG, loxP-flanked
enhanced green fluorescent protein (EGFP)
cDNA/chloramphenicol acetyltransferase (CAT)
gene, and the lacZ gene (encoding the βgalactosidase), and are used for lineage-analysis in
mice with the Cre-loxP system [15]. Briefly,
solutions containing circular pCAG/NCre
plasmid/lipid complexes were prepared at
volumes of 70 µl/testis. For each solution, 16 µl of
FuGENE™6 (Boehringer Mannheim GmbH,
Mannheim, Germany) was diluted with 24 µl of
phosphate-buffered saline without Ca2+ and Mg2+
[PBS(-)], pH 7.2, and then added to 8 µg of
plasmid DNA dissolved in PBS(-) according to the
manufacturer’s protocol. The resulting DNA/lipid
complex solution was slowly injected into the
testis of CETZ-17 transgenic male with a 30guage needle (Natsume, Tokyo, Japan) attached
to a 1-ml plastic disposable syringe (Termo,
Tokyo, Japan) at a depth of 5-6 mm through the
capsule of the testis. After injection, the needle
was slowly removed. Both testes were injected.
For multiple injections (3 repeated injections),
injection was repeated 3 days apart. At 7 to 21
days after the final injection, the injected males
were mated to superovulated CETZ-17 females
every 2 days to obtain F0 pups. These CETZ-17
females had previously been induced to
superovulate by two gonadotrophin (eCG-hCG)
treatments spaced about 2 days apart.
At weaning stage, these F0 mice were subjected
to tail isolation for genotyping, and the mice
identified as those carrying transgenes were next
mated to normal B6C3F1 mice (purchased from
Clea Japan, Inc., Tokyo, Japan) to obtain F1
offspring. F2 offspring were also obtained from
mating between transgenic F1 mice and normal
B6C31 mice.
These mice were kept on a 12 h light/12 h dark
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124
schedule (lights on from 0700 h to 1900 h) and
allowed food and water ad libitum. Experiments
were carried out in accordance with the Guide for
the Care and Use of Laboratory Animals of Tokai
University.
Isolation of Tissues and Genomic Southern
Analysis
Genomic DNA of tails from weaned mice was
isolated as previously described [17] with several
modifications [9]. Genomic DNA (10 µg) was
digested with Eco RV and Bgl II, which cleave
pCAG/NCre DNA twice and release an
approximately 1.0-kb fragment containing a 3’
portion of NCre gene, and fractionated by 0.8%
agarose gel electrophoresis. To obtain a copy
control, 1.17 pg of pCAG/NCre DNA was
calculated to be equivalent to one copy of the
DNA per diploid cell, based on a genome size of 6
9
x 10 bp per diploid cell. Based on this
calculation, purified pCAG/NCre DNA was
added to mouse tail (non-treated) DNA to obtain
various copy levels of pCAG/NCre DNA, and
processed concomitantly as for the experimental
samples. After electrophoresis, the DNA was
transferred to nylon membrane filters
(GeneScreenPlus; NEN, Boston, MA). The filters
were hybridized as described by Sato et al. [9]. An
Eco RV and Bgl II 0.71-kb fragment (hereafter
32
termed NCre probe) of pCAG/NCre was Plabelled by a random priming labelling method
[18] with an Amersham multiprime labelling kit
(Amersham, Buckinghamshire, England) and used
as a probe.
PCR Analysis
PCR amplification reactions were performed in
a total volume of 10 µl, containing 10 mM TrisHCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.25
mM each of dATP, dCTP, dGTP and dTTP, 1 mM
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S AT O A N D N A K A M U R A
primers, 1 µg genomic DNA and 0.5 units Taq
polymerase (TaKaRa Taq™; #R001A, Takara
Shuzo Co., Ltd., Tokyo, Japan), as described
previously [19]. Forty cycles of PCR reactions
were performed in a Perkin Elmer DNA Thermal
Cycler (480) with cycle times of 1 min at 94˚C, 1
min at 56˚C and 4 min at 72˚C.
Eight sets of primers (A to H) for detection of
the introduced pCAG/NCre DNA and the
expected length of the DNA fragments amplified
by each primer set are shown in Figure 1. For the
first screening of tail samples obtained after mating
with testis-injected males, primer set A (Cre-2S and
Cre-2RV) was used. This primer set yields 300-bp
fragments from the 3’ portion of the NCre gene.
Cre-2S (5’-GAT CCG AAT AAC TAC CTG TTT3’) corresponds to nucleotides 1,441 to 1,461 in
the Cre gene sequence [20], and Cre-2RV (5’-TGT
TTC ACT ATC CAG GTT ACG-3’) corresponds
to nucleotides 1,740 to 1,720 in the Cre gene
sequence [20]. For amplification of a region
corresponding to portions of pBR322 and
pBluescript SK(-), primer set B (BR-S and Ori-S)
was used. This primer set yields approximately 0.8kb fragments. BR-S (5’-CTG CTT CCT AAT GCA
GGA GTC-3’) corresponds to nucleotides 616 to
636 in the pBR322 sequence [21], and Ori-S (5’AAC TGA GAT ACC TAC AGC GTG-3’)
corresponds to nucleotides 1,433 to 1,413 in the
pBluescript SK(-) sequence (Accession No.
X52330). For amplification of a region
corresponding to a portion (containing a portion of
ampicillin resistance gene) of pBluescript SK(-),
primer set C (Amp-S and Ori-RV) was used. This
primer set yields approximately 1.1-kb fragments.
Amp-S (5’-TTG AGT ACT CAC CAG TCA CAG3’) corresponds to nucleotides 2,532 to 2,512 in
the pBluescript sequence, and Ori-RV (5’-CAC
GCT GTA GGT ATC TCA GTT-3’) corresponds
to nucleotides 1,414 to 1,434 in the pBluescript
SK(-) sequence. For amplification of a portion of
cyomegalovirus ehancer and an ampicillin
T E S T I S - M E D I AT E D G E N E T R A N S F E R ( T M G T )
resistance gene-containing region in pBluescript
SK(-), primer set D (Amp-RV and CME-RV) was
used. This primer set yields approximately 1.55-kb
fragments. Amp-RV (5’-GGC TCC AGA TTT
ATC AGC AAT -3’) corresponds to nucleotides
2,129 to 2,149 in the pBluescript sequence, and
CME-RV (5’-ATG GGC TAT GAA CTA ATG
ACC-3’) corresponds to nucleotides 202 to 182 in
the cytomegalovirus enhancer sequence [22]. For
amplification of a region corresponding to the 3rd
exon of rabbit β-globine gene and the 5’ region of
NCre gene in the CAG-NCre insert, primer set E
(β-gl-1 and Cre-RV) was used. This primer set
yields 381-bp fragments from the 5’ portion of the
Cre gene in pCAG/NCre. β-gl-1 (5’-CTC CTG
GGC AAC GTG CTG GT-3’; [23]) corresponds to
the 3rd exon of the rabbit β-globin gene sequence
[24] from nucleotides 1,068 to 1,087, and Cre-RV
(5’-ATG AAG CAT GTT TAG CTG GCC-3’; [25])
corresponds to nucleotides 761 to 781 in the 5’
region of the Cre gene sequence [20]. For
amplification of a region corresponding to the 1st
intron of chicken β-actin gene, and a portion of the
2nd intron and 3rd exon of rabbit β-globin gene in
the CAG-NCre insert, primer set F (βA-1 and Ex3RV) was used. This primer set yields approximately
520-bp fragments. βA-1 (5’-TCT GAC TGA CCG
CGT TAC TCC CAC A-3’) corresponds to
nucleotides -1,011 to –987 of the chicken β-actin
gene sequence [26], and Ex3-RV (5’-AAC CAG
CAC GTT GCC CAG GAG-3’) corresponds to
nucleotides 1,311 to 1,291 in the 3rd exon of the
rabbit β-globin gene sequence [26]. For
amplification of a region corresponding to the 3’
region of NCre gene and a portion of 3’-noncoding
region of the rabbit β-globin gene, primer set G
(Cre-2S and BGL-RV) was used. This primer set
yields approximately 0.8-kb fragments. BGL-RV
(5’-GCC AGA AGT CAG ATG CTC AAG-3’)
corresponds to nucleotides 1,485 to 1,465 in the
3’-noncoding region of the rabbit β-globin gene
sequence [24]. For amplification of a region
125
corresponding to the 3’ region of the NCre gene,
the 3’-noncoding region of rabbit β-globin gene,
and SV40 poly(A) signals in the CAG-NCre insert,
primer set H (Cre-2S and SV-RV) was used. This
primer set yields approximately 1.2-kb fragments.
SV-RV (5’-ACA ACT AGA ATG CAG TGA AAA3’) corresponds to nucleotides 2,557 to 2,577 in
the poly(A) signals of the SV40 DNA sequence
[27]. As a positive control, 5 ng of pCAG/NCre
DNA was used. As a negative control, 1 µg of
genomic tail DNA from a non-transgenic mice was
used. Precautionary measures were taken to
minimise contamination; PCR mixes were
prepared in a biological containment cabinet using
sterilized pipette tips in a separate room from
where reactions, storage, and handling of PCR
products were performed. A separate pipetters and
aerosol-resistant tips were used to add PCR premix
to the samples.
Products of the reaction were analyzed by
electrophoresis on a 2% agarose gel. A 100 bp
ladder (Promega Co., Madison, MI) was used as a
molecular weight marker. The gels were stained
with ethidium bromide (EtBr), and the amplified
DNA bands were visualized by UV illumination.
Quantitation of pCAG/NCre DNA in
Fetal Samples by PCR
To assess the quantity of exogenous DNA in
the TMGT-derived offspring, this DNA was used
for PCR templates. By comparing the PCR
products with PCR products from the copy
controls, we calculated the amount of exogenous
DNA present. For copy number controls, one
copy of pCAG/NCre could be calculated when
1.17 pg of pCAG/NCre DNA was contained in 1
µg of mouse genomic DNA, as described
previously. Based on this calculation, 1.17 pg of
purified pCAG/NCre DNA was diluted with a
solution containing 1 µg of genomic tail DNA
from non-transgenic mouse to obtain 1 copy of
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S AT O A N D N A K A M U R A
FIGURE 1
Structure of approximately 7-kb pCAG/NCre. The small arrows indicate the positions and directions of the primers used here. The
solid lines above pCAG/NCre construct indicate the regions PCR-amplified. The bold line indicates a portion of the 2nd intron,
3rd exon and 3’-noncoding region of the rabbit β-globin gene. The dashed line indicates a portion of pBR322. The thin line
indicates pBluescript SK(-) vector. AmpR, ampicillin resistance gene; ATG, translation initiation site; CAG, cytomaglovirus
enhancer + chicken β-actin promoter; NCre, nuclear location signal + Cre gene; ori, replication origin of pBluescript SK(-) vector;
SVpA, polyadenylation sites of SV40 gene.
pCAG/NCre DNA, and then serially diluted with
water 10-104-fold. DNA samples from tails were
first prepared at a concentration of 1 µg/µl, and
then serially diluted with water 10-10 4 -fold.
Thirty cycles of PCR with the primer set A (Cre2S and Cre-2RV) were performed on the samples
with cycle times of 1 min at 94˚C, 1 min at 56˚C
and 2 min at 72˚C. Products of the reaction were
analyzed by electrophoresis on a 2% agarose gel.
The gels were stained with EtBr and the amplified
DNA bands were visualized by UV illumination.
TRANSGENICS
Isolation of PCR Products and Subcloning
Into TA Cloning Vector for Sequencing
Tail DNA was PCR-amplified using the same
conditions as for detection of each part of
pCAG/NCre plasmid, except that the total volume
was increased to 100 µl. The PCR products were
ethanol-precipitated and then electrophoresed
through a 1% low-melting-temperature agarose gel
(SeaPlaque GTG Agarose; FMC BioProducts,
Rockland, ME). The gel slice containing the PCR
T E S T I S - M E D I AT E D G E N E T R A N S F E R ( T M G T )
127
FIGURE 2
A. PCR analysis of genomic DNA of F0 mouse tail. PCR reaction using a primer set A (Cre-2S and Cre-2RV) yielded a 300-bp
product (indicated by arrows). Staining of gels with EtBr is shown. Samples indicated by * and ** above the lanes were from
females mated to DNA-injected males #2 and #4, respectively. C, non-transgenic tail DNA as negative control; PC, 5 ng of
pCAG/NCre as positive control. “m” indicates 100-bp ladder markers. B. Genomic Southern blot analysis of randomly selected
F0 mouse tail samples (lanes 1 to 8) derived from DNA-injected male #1 that had been identified as NCre gene positive using
primer set A. Genomic DNA (10 µg/lane) was digested with Eco RV/Bgl II (which releases an approximately 0.71-kb fragment
containing a 3’ portion of NCre gene) and blotted onto nylon filters prior to hybridization with NCre probe. Each experimental
sample was negative for the probe. The copy control C0.1 which corresponds to 0.1 copies/sample did not exhibit any detectable
band, while the other copy controls C1, C2 and C5, corresponding to 1, 2 and 5 copies/sample, respectively, exhibited detectable
band of expected size (indicated by an arrow). C, DNA from non-transgenic tail. C. Quantitation of pCAG/NCre DNA in TMGTderived F0 tails. Genomic DNA samples were first prepared at 1 µg/µl, and then serially diluted with water 10-104 fold. For copy
number controls, purified vector DNA (1.17 pg) was dissolved in a solution containing 1 µg genomic DNA, which yielded 1 copy
of DNA per diploid cell. This solution was then serially diluted with water 10-104 fold. These genomic DNA samples and copy
number controls were then subjected to PCR using primer set A. All TMGT-derived samples (including samples at F0 to F2
generations) were judged as those with less than 1 copy of exogenous DNA per diploid cell, since they exhibited rapid reduction
in intensity of the target band after 10 to 102-fold dilution, whereas the copy control samples did not.
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S AT O A N D N A K A M U R A
TABLE I
Gene transmission to F0 offspring in the TMGT system and the ratio of mice carrying the intact
pCAG/NCre plasmid exogenously introduced.
Males
testis-injected
No. of F0
mice obtained
No. of mice positive
for the presence
of NCre gene (%)a
No. of mice
carrying intact
pCAG/NCre (%)b
#1
15
13 (87)
7 (54)
#2
17
14 (82)
10 (71)
#3
23
23 (100)
12 (50)
#4
12
9 (75)
5 (56)
a
For detection of 3’ portion of NCre gene in pCAG/NCre, genomic DNA was PCR-amplified using primer set A (see Figure 1).
Samples carrying 300-bp band were considered positive for the presence of NCre gene.
b
For detection of intact type of pCAG/NCre in the TMGT-derived samples, PCR was performed using primer set B (see Figure 1).
Samples carrying an expected band of approximately 0.8-kb were considered those carrying intact pCAG/NCre.
products was melted at 65°C for 30 min and then
extracted by saturated phenol. After precipitation
with ethanol, the gel-purified PCR product was
ligated to TA cloning vector pCR™2.1 (Invitrogen
Co., Carlsbad, CA) and the resulting recombinants
were evaluated for automated fluorescent
sequencing analysis using the ABI PRISM™ Dye
Terminator Cycle Sequencing Ready Reaction Kit
with AmpliTaq DNA polymerase, FS (PE Applied
Biosystems, Foster City, CA), and universal primer.
size detectable after staining with EtBr of gels
(Table I). An example of PCR profiles is shown
in Figure 2A. However, none of these
pCAG/NCre-positive samples possessed more
than 1 copy of pCAG/NCre per diploid cell,
since genomic Southern blot analysis of these
PCR-positive samples failed to detect any
hybridizable band despite repeated trials. An
example of genomic Southern blot hybridization
is shown in Figure 2B.
RESULTS
Quantitation of pCAG/NCre DNA in Tail
Samples by PCR
Identification of pCAG/NCre DNA in F0
Offspring Obtained From Mating With
DNA-Injected Males
On PCR analysis for detection of pCAG/NCre
plasmid using primer set A (Figure 1) of genomic
DNA isolated from a total of 67 F0 tails from
females that had been mated to 4 DNA-injected
males (termed #1 to #4), 75 to 100% of the
samples had amplified products of the expected
TRANSGENICS
To assess the quantity of foreign DNA in these
PCR-positive samples, the tail DNA samples (1
µg/µl; corresponding to lanes 1 to 8 in Figure 2B)
were serially diluted with water 10-10 4 -fold.
Concomitantly, 1.17 pg of purified pCAG/NCre
DNA was first diluted with a solution containing
1 µg of genomic DNA from non-transgenic tail,
and then serially diluted with water 10-104-fold.
These diluted samples were subjected to PCR
using primer set A. The results are shown in
T E S T I S - M E D I AT E D G E N E T R A N S F E R ( T M G T )
129
FIGURE 3
PCR analyses of randomly selected F0 mouse tail samples (lanes 1 to 8 in Figure 2B) derived from male #1 using primer sets A to
C and E to H. Arrows indicate the expected band for each reaction. C, non-transgenic tail DNA as negative control; PC, 5 ng of
pCAG/NCre as positive control. “m” indicates 100-bp ladder markers
Figure 2C. As expected, all F0 samples (in which
three samples are shown as an example in Figure
2C) were estimated to have far less than 1 copy
of pCAG/NCre, although slight variation in the
level of pCAG/NCre was seen through F0 tail
DNA samples.
At Least Two Types of pCAG/NCre Exist in
the F0 Offspring Obtained After TMGT
We examined whether the exogenous
pCAG/NCre DNA transmitted to F0 offspring
obtained after TMGT exists as an intact form. We
first performed long distance (LD) PCR [28] to
amplify the entire length of pCAG/NCre
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S AT O A N D N A K A M U R A
FIGURE 4
Pedegree of DNA-injected male #1. Transgenicity was first evaluated for tail DNA by PCR using primer set A. Of the NCre genepositive samples, second screening with PCR was performed using primer set B. Mice which were negative for the presence of NCre
gene at first screening are designated non-transgenic and indicated by white boxes. Mice which appeared to possess intact
pCAG/NCre after second screening are indicated by solid boxes, while mice which appeared to possess deleted form of pCAG/NCre
are indicated by dashed boxes. Squares and circles indicate males and females, respectively. Mice are numbered below each symbol.
(approximately 7 kb in size) for genomic DNA
isolated from F0 tails that has been identified for
the presence of the 3’ region of the NCre gene by
PCR with primer set A. Unfortunately, this trial
failed, probably because the copy number of the
introduced pCAG/NCre was very low, as
mentioned above. We thus decided to perform
general PCR using three other primer sets (B-D;
Figure 1) recognizing different portions of
pBluescript SK(-) vector, a plasmid backbone for
pCAG/NCre. With these primer sets, DNA
fragments ranging from 0.8 to 1.55 kb could
easily be amplified. When 8 F0 tail samples,
which were the same used for genomic Southern
blot analysis (Figure 2B) and had been identified
as NCre gene-positive using primer set A (panel A
in Figure 3), were subjected to PCR amplification
using primer set B, only two samples (lanes 1 and
2 in the panel B of Figure 3) yielded a band of the
TRANSGENICS
expected size (approximately 0.8 kb), and the
remaining 6 samples were completely negative for
this band (lanes 3-8 in the panel B of Figure 3).
The same samples were next subjected to PCR
using primer set C. As shown in the panel C of
Figure 3, only two samples corresponding to lanes
1 and 2 possessed the expected 1.1-kb band,
while the remaining ones did not. PCR using
primer set D yielded the same results as obtained
from PCR using primer sets B and C (data not
shown). These findings suggest that the samples
corresponding to lanes 3-8 in Figure 3 contain a
portion of the NCre gene, but lack a portion
corresponding to the pBluescript SK(-) vector
sequence. On the other hand, the samples
corresponding to lanes 1 and 2 in Figure 3 appear
to contain all of pCAG/NCre.
Sequencing of the products PCR-amplified
from the sample corresponding to lane 1 in Figure
T E S T I S - M E D I AT E D G E N E T R A N S F E R ( T M G T )
131
FIGURE 5
PCR analysis of F1 and F2 tail DNA samples derived from lines #9 and #10 using primer set B. All samples positive for the
presence of the 3’ portion of NCre gene after PCR using primer set A were tested. Arrows indicate the band as expected. Circles
above lanes indicate samples which appear to carry intact pCAG/NCre in their genome. The number for each lane corresponds to
mice shown in Figure 4. PC, 5 ng of pCAG/NCre as positive control. “m” indicates 100-bp ladder markers.
3 using primer sets A to D demonstrated that
sequence homologies between the PCR products
and authentic plasmid were 100, 99.5, 100 and
99.3% for the regions corresponding to A to D in
pCAG/NCre, respectively (data not shown). This
comfirmed that the introduced pCAG/NCre DNA
in the sample corresponding to lane 1 in Figure 3
was present in intact form. From these findings, it
may be concluded that there are at least two types
of pCAG/NCre, an intact type and a deleted one,
in TMGT-derived F0 mice.
We next determined the percentage of intact
pCAG/NCre present in the F0 offspring obtained
from mating with each of 4 DNA-injected males.
Screening was performed using PCR with primer
set B for the tail DNA samples that had
previously been identified as transgenics using
primer set A. As listed in Table I, 50-71% of the
F0 offspring appeared to possess intact
pCAG/NCre in their genome.
An Attempt to Determine Deleted Regions
in pCAG/NCre in the TMGT-Derived F0
Offspring
As previously described, we found that the
introduced pCAG/NCre plasmid lacked the
pBluescript SK(-) vector sequence in 6 of 8 NCre
gene-positive tails tested. In an attempt to
determine the deletion sites in pCAG/NCre in
more detail, we performed PCR using primer sets
E to H (Figure 1). When the same samples shown
in the panels A to C in Figure 3 were PCRamplified using primer sets E and G, which
recognize the 5’ and 3’ end regions of the NCre
gene in pCAG/NCre, respectively, a distinct band
of the expected size was observed in all the
samples tested for each PCR (panels E and G in
Figure 3). PCR using primer sets F and H,
however, failed to generate a distinct band in the
6 samples (corresponding to lanes 3 to 8 in Figure
TRANSGENICS
132
S AT O A N D N A K A M U R A
3) considered to carry a deleted form of
pCAG/NCre. The remaining two samples
(corresponding to lanes 1 and 2 in Figure 3),
which had been considered to possess intact
pCAG/NCre, exhibited a distinct band of the
expected size. These findings suggest that deletion
may have occurred at a portion spanning CAG to
the 1st intron of the chicken β-actin gene and a
portion spanning a 3’-noncoding region of the
rabbit β-globin gene to SV40 poly(A) signals in
pCAG/NCre.
was found in any F2 offspring obtained after
mating between the F1 mouse carrying deleted
form of pCAG/NCre and a normal mouse (Figure
4; lower panel of Figure 5), suggesting that once
deletion occurs, recovery of intact pCAG/NCre is
impossible.
PCR-mediated quantitation of pCAG/NCre
DNA in the F1 and F2 tail samples revealed that
these F1 and F2 samples had far less than 1 copy of
pCAG/NCre as did F0 parental sample (Figure 2C).
Pattern of Transmission of pCAG/NCre
From F0 to F2 Generations
DISCUSSION
To examine the pattern of transmission of the
exogenous plasmid pCAG/NCre DNA in the
TMGT-derived mice, we made two lines (termed
“line #9” and “line #10”) derived from the DNAinjected male #1. In Figure 4, the pedgrees of lines
#9 and #10 are shown. The parental F0 females
(#9 and #10) were previously identified as those
carrying intact pCAG/NCre after PCR using
primer sets A and B. Tail DNA samples from the
F1 and F2 generations were first examined for the
presence of 3’ portion of NCre gene by PCR using
primer set A. As shown in Figure 4, high rates of
gene transmission were obtained from F0 to F1
and F1 to F2 generations in this TMGT system, as
pointed out previously [12].
The 2nd genotyping using PCR with primer set
B was next performed to identify mice carrying
intact pCAG/NCre. Figure 5 indicates the results
of genotyping of samples from the F1 and F2
generations derived from lines #9 and #10
obtained by PCR using primer set B. The intact
pCAG/NCre present in both parental F0 females
(#9 and #10) was not transmitted to all F1
offspring. In other words, deletion of
pCAG/NCre occurred during transition from the
F0 to F1 generations with a relatively high degree
of frequency. Interestingly, no intact pCAG/NCre
TRANSGENICS
In this study, we used CETZ-17 transgenic mice
[15] to visualize the effect of transgene expression
of a Cre expression plasmid, pCAG/NCre. The
timing and extent of expression of exogenous
DNA can be studied using CETZ-17 mice, since
transient Cre expression will leave behind a
permanent signature in the form of lacZ activity. In
a preliminary test [29], we observed that
expression of pCAG/NCre could be detected by
RT-PCR, although it appeared to be very weak,
and the number of fetuses expressing NCre mRNA
was low (24%) among those carrying exogenous
DNA. We found that 14.9% of TMGT-derived
mid-gestational F0 fetuses exhibited lacZ activity,
although strength of activity was very low. This
efficiency (14.9%) of fetal expression of lacZ
activity appears to be roughly correlated with the
efficiency (24%) of expression of NCre mRNA by
fetuses. Furthermore, we observed production of
CZ, a recombined fragment generated from the
integrated CETZ-17 transgenes, in the NCre
mRNA-expressing fetuses by PCR. Unfortunately,
this NCre mRNA expression and generation of CZ
could not be detected at F1 and F2 generations
(data not shown). These findings suggest that
expression of the exogenous gene is transient,
probably because pCAG/NCre is unintegrated.
One of the most important issues to be
T E S T I S - M E D I AT E D G E N E T R A N S F E R ( T M G T )
considered in the TMGT system appears to be
how the exogenous plasmid DNA is transmitted
from one generation to the next and is present in
mouse tissues. As mentioned above, the TMGT
system differs from pronuclear microinjectionmediated transgenesis in several respect such as
difficulty in detection of hybridizable bands on
genomic Southern blot hybridization, lack of
success in plasmid rescue experiments, high
frequency of generation of transgenic progeny,
inability to introduce more than 1 copy of
exogenous plasmid DNA per diploid cell into
TMGT-derived offspring and high frequency
(non-Mendelian) of transmission of the transgene
from one generation to the next. DNA may thus
be present mosaically in tissues. Mosaicism
should not be passed on to the next generation.
At present, it is difficult to imagine a mechanism
by which epigenetic genes could be passed on.
At present, we are unable to explain why or
how the foreign DNA sequences are propagated
in growing fetuses and probably in adult mouse
tissues, since the replication origin of pCAG/NCre
plasmid (with pBluescript SK(-) backbone) is
derived from E. coli. In this study, we
demonstrated that exogenous pCAG/NCre
plasmid is present intact in tissues of the TMGTderived F0 offspring, suggesting that pCAG/NCre
may be maintained extrachromosomally. To
confirm this, we performed plasmid rescue [30] or
Hirt extraction [31] or a modification of it [32] to
recover pCAG/NCre plasmid from the F0 tail
samples carrying intact pCAG/NCre, but both
failed despite repeated trials (data not shown). We
also performed inverse PCR [33,34] to test for
integration of the foreign sequences into host
genome. Unfortunately, we were unable to detect
any chromosomal sequences flanked by the
transgene (data not shown). Further
modifications and refinements will be required to
determine whether the exogenous plasmid DNA
is present episomally or integrated into the host
133
genome. One possible approach would be
construction of a genomic library from tail DNA
of the TMGT-derived F0 or F1 offspring. With
this library, genomic clones carrying a
chromosomal sequence flanked by the transgene
will be picked up if the transgene is integrated
into the mouse genome. Another approach is to
introduce a DNA fragment having no replication
origin or linearized plasmid DNA into testes. In
the former case, the DNA fragment lacking a
replication origin would exhibit different
behavior, as does plasmid DNA in the TMGT
system. In the latter case, when the linearized
plasmid DNA is introduced into testes, it might be
recircularized in mouse tissues and then behave
like a circular plasmid.
Another interesting finding of this study is the
generation of a deleted form of the plasmid
exogenously introduced. Interestingly, the
deletion sites in pCAG/NCre plasmid appeared to
be the same among the F0 tail samples tested (see
Figure 3). These deletions were confined to two
regions (a portion spanning CAG to the 1st intron
of the chicken β-actin gene and a portion
spanning the 3’-noncoding region of the rabbit βglobin gene to SV40 poly(A) signals) in
pCAG/NCre. Furthermore, this type of deletion
was also found in F1 offspring (see Figures 4,5).
Once deletion occurs, recovery of intact
pCAG/NCre appears to be difficult, since mating
of F1 mice carrying a deleted form of
pCAG/NCre with normal mice always resulted in
production of F2 offspring carrying a deleted
form of pCAG/NCre (see Figure 4). These
findings suggest the presence of a mechanism of
regulation to elicit deletion of the exogenous
plasmid pCAG/NCre in mice. To test this,
detailed mapping of the deletion sites found in the
TMGT-derived offspring is now ongoing.
Acknowledgments
We thank Dr. Toshiteru Watanabe (Tokai
TRANSGENICS
134
University School of Medicine) for maintaining
DNA-injected animals and providing them to us.
We are also grateful to Drs. Minoru Kimura
(Tokai University School of Medicine), Shyoso
Ogawa (Meiji University) and Norihiro Tada
(Juntendo University School of Medicine) for
their helpful comments and suggestions on the
TMGT technology. This study was supported by
a grant from The Ministry of Education, Science,
Sports and Culture, Japan.
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