Exp. Anim. 57(5), 471–477, 2008

—Note—

Establishing EGFP Congenic Mice in a NOD/Shi-scid
IL2Rgnull (NOG) Genetic Background Using aMarker-Assisted Selection Protocol (MASP)
Hiroshi SUEMIZU, Chie YAGIHASHI, Tomoko MIZUSHIMA, Tomoyuki OGURA,
Tomoo ETOH, Kenji KAWAI, and Mamoru ITO
Central Institute for Experimental Animals, 1430 Nogawa, Miyamae, Kawasaki 216-0001, Japan
Abstract: Severely immunocompromised NOD/Shi-scid IL2Rgnull (NOG) mice, which show
higher engraftment efficiency, are useful as recipients in xenotransplantation studies. We
generated a NOG-enhanced green fluorescent protein (EGFP) transgenic (Tg) mouse (NOGEGFP) that was introduced the EGFP transgene from the C57BL/6-EGFP Tg mouse using
the speed congenic method with a marker-assisted selection protocol (MASP). With thismethod, the selection of the male with the closest NOG strain type was repeated four times.
When human cord blood CD34+ cells were transplanted into NOD/Shi-scid, NOG, and NOGEGFP mice (N6), the engraftment efficiency of the NOG-EGFP mice was significantly higher
than that of the NOD/Shi-scid mice and was similar to that of the NOG mice. These results
suggest that the NOG-EGFP mice, which were generated using the congenic method with
MASP, acquired the immunological properties of the NOG mice.
Key words: microsatellite marker, NOG mouse, speed congenic

Previously established techniques of introducing additional genetic changes into transgenic or knockout
mice include transgenic methods involving the direct
microinjection of fertilized eggs and congenic strategies
using extant genetically modified mice. The congenic
strategy is the classical method and is easiest to perform,
but, backcrossing must be performed for at least seven
generations to replace over 99% of the genetic background (99.2% is theoretically replaced), which is extremely time-consuming. One of the fastest ways tointroduce additional genetic modification is the markerassisted selection protocol (MASP) known as the “speed
congenic” method. In MASP, the male mouse showing

the most complete replacement with the targeted genetic background is selected as the “best” male and isused in the next backcross [11–13]. NOG (formally,
NOD.Cg-Prkdcscid Il2rgtm1Sug/ShiJic) mice, which weredeveloped by introducing IL2Rg null mutation from
C57BL/6-Il2rg tm1Sug mice with backcross mating to
NOD/Shi-scid mice [3], have no lymphocytes (neither
T nor B) or natural killer (NK) cells, and have impaired
dendritic cell function [4, 7]. Therefore, NOG mice canbe used to develop “humanized mice”, which possesshigh levels of human-derived cells or tissues. Because
it is difficult to label human cells (e.g. hematopoietic
stem cells or neural stem cells) with visible markers, we(Received 26 July 2007 / Accepted 21 April 2008)
Address corresponding: H. Suemizu, Central Institute for Experimental Animals, 1430 Nogawa, Miyamae, Kawasaki 216-0001, Japan

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H. Suemizu, ET AL.

attempted to introduce the enhanced green fluorescentprotein (EGFP) gene into the NOG mouse as a visible
recipient marker, which if successful would make iteasier to differentiate donor cells from recipient cells instudies of transplantation and regenerative medicine.
This study was performed in accordance with institutional guidelines and was approved by the Animal Experimentation Committee of the Central Institute forExperimental Animals. NOG mice expressing the EGFP
gene can be obtained reliably by backcrossing with ananimal in which the phenotype of the modified gene isalready expressed in the donor. Therefore, we mated an
inbred line of C57BL/6-Tg(Act-EGFP)C14-Y01FM1310sb (C57BL/6-TgEGFP) mice [6, 8] with a NOG
mice to make a NOG-EGFP (NOD.Cg-Prkdcscid Il2rgtm1Sug
Tg(Act-EGFP)C14-Y01-FM1310sb/ShiJic) mice. Tailclips of the mice were obtained and digested with proteinase K using standard methods [9]. Genomic DNA
was extracted using the MagExtractor System MFX9600 Magnia R Plus (Toyobo, Osaka, Japan) according
to the manufacturer’s instructions. The EGFP transgene
was genotyped by PCR with the forward primer GFP-F1(5’-CTGGTCGAGCTGGACGGCGACG-3’) and reverse primer GFP-R1 (5’-CACGAACTCCAGCAGGACCATG-3’). The scid and IL2Rgnull mutations were
genotyped using a previously described PCR method [2,3]. Three to six microsatellite markers were selectedfrom each chromosome, including the X-chromosome.
We selected 87 microsatellite markers to evaluate themouse genetic backgrounds of the C57BL/6, NOG, and
129S6/SvEv strains because the Tg EGFP mice originated in C57BL/6, and the IL2Rgnull mutants were generated with mouse ES cells (CCE) derived from the
129S6/SvEv strain. To evaluate the genetic backgroundin more detail, an additional 17 microsatellite markers
were arranged on chromosome 9. General information
about the primers, including the size of the PCR product,
is listed in Table 1. First, 10–100 ng of genomic DNA
was suspended in a total volume of 12.5 µl PCR buffer
containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5mM MgCl2, 0.1–0.3 µM fluorescence-labeled primers,
0.2 mM dNTP, and 1.0 unit of Platinum GenoType Tsp
DNA polymerase (Invitrogen, Carlsbad, CA, USA). Using a Bio-Rad iCycler (Bio-Rad Laboratories, Hercules,
CA, USA), the thermal cycling conditions consisted of

1 cycle at 95°C for 2 min followed by: 10 cycles at 94°C
for 45 s, 60°C for 1 min, and 72°C for 1 min; then, 17
cycles at 89°C for 1 min, 60°C for 1 min, and 72°C for
1 min; with a final extension of 10 min at 72°C. Following PCR, 2 µl of product, 0.6 µl of GS500-LIZ size
standard (Applied Biosystems, Foster City, CA, USA),and 24.4 µl of Hi-Di formamide (Applied Biosystems)
were mixed and denatured at 95°C for 2 min, cooled onice, and then loaded directly on an ABI 3130xl Genetic
Analyzer (Applied Biosystems). The electrophoresis
data were processed using GeneMapper 4.0 software(Applied Biosystems). When non-labeled primer pairs
were used, the PCR products were electrophoresed on
3% NuSieve 3:1 agarose (Lonza Walkersville, Inc.,
Walkersville, MD, USA) gel/20 mM Tris-acetate, 2.5
mM EDTA (0.5 × TAE) and then stained with ethidium
bromide (0.25 µg/ml). Statistical analyses were performed with StatView 5.0 and Prism 5 software.
To introduce the EGFP transgene into NOG mice, thefirst generation hybrid (F1) was obtained by mating amale C57BL/6-TgEGFP mouse as the donor with a recipient NOG female mouse. The F1 hybrid received auniform genome (except for the sex chromosome) fromthe donor and recipient, and the replacement rate (described as “% Recipient genome” in Table 2) for boththe expected and observed values was 50% (Table 2).
In the next stage, a randomly selected male F1 mousewith the EGFP transgene was again mated with female
NOG recipient mice to obtain the N2 generation. Marker-assisted selection was started at the N2 generation to
select the “best” male congenic mouse. Using the 61
informative markers, the male with the closest NOG
strain type was selected as the parent for the N3 generation. Using this method, the selection of males with theclosest NOG strain type was repeated four times. The
observed “% Recipient genome” of the male closest to
the NOG background was 81.1, 95.9, 98.4, and 99.2%
in the N2, N3, N4, and N5 generations, respectively. The
higher values of the observed “% Recipient genome” ingeneration N 3 and N 4 were statistically significant(P=0.018 and 0.067, respectively, Fisher’s exact probability test). Wakeland et al. described the rationale ofspeed congenics, and stated that the advantage of screening with low-density markers (25 cM marker spacing)
was realized in the N3 and N4 generations [12]. Com-

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Generation of NOG-EGFP mice
Table 1. MASP primer pairs and PCR product sizes in the NOG, C57BL/6, and 129S6/SvEv strains
Marker
D1Mit67 a
D1Mit303
D1Mit132 a
D1Mit91
D1Mit102 a
D1Mit459 a
D2Mit1 a
D2Mit312
D2Mit182 a
D2Mit311 a
D2Mit346 a
D3Mit149 *a
D3Mit62
D3Mit25 a
D3Mit85 a
D3Mit89 a
D4Mit227 a
D4Mit52 a
D4Mit190
D4Mit256 a
D5Mit146 a
D5Mit1
D5Mit58 a
D5Mit367 a
D5Mit97 a
D6Mit86 a
D6Mit284 a
D6Mit304 a
D7Mit267 a
D7Mit193
D7Mit350 a
D7Mit100
D7Mit189 a
D8Mit155 *a
D8Mit217
D8Mit191 a
D8Mit88 a
D8Mit93
D9Mit250
D9Mit83 a
D9Mit97 a
D9Mit102
D9Mit207
D9Mit105
D9Mit259
D9Mit107
D9Mit8
D9Mit124
D9Mit236
D9Mit11
D9Mit275
D9Mit35

Position
129S6/SvEv
NOG C57BL/6
Dye
Marker(cM) b			
(CCE ES)			
9
34.8
43.1
64
73
102
1
1
38.3
83.1
91.8
2.4
4.6
29.5
72.9
86.1
3.2
54.9
79
82.7
1
5
41
65
74
0.5
37.5
75
11
24.5
41
53.5
72.4
1
6
21
58
72
5
6
29
31
33
35
38
40
42
42
43
48
50
52

125
112
160
146
121
116
111
120
149
110
107
147
117
127
226
214
182
110
ND
128
125
135
116
96
118
121
129
105
174
149
138
201
114
156
169
122
127
169
123
129
162
144
160
116
122
119
185
128
125
112
115
112

133
122
141
146
110
120
115
120
153
121
101
138
117
129
218
220
180
119
138
132
120
135
114
102
124
132
138
115
187
149
116
201
129
161
169
133
114
169
123
134
151
140
148
145
112
120
178
124
143
74
110
124

125
112
160
138
125
116
113
111
149
115
118
138
110
123
224
214
172
112
130
128
115
129
123
92
118
121
129
107
199
159
122
193
114
108
179
122
114
163
132
134
159
144
155
145
115
104
169
124
125
100
110
124

NED
NED
NED
PET
FAM
PET
VIC
PET
PET
VIC
PET
PET
NED
VIC
VIC
FAM
VIC
PET
FAM
FAM
PET
VIC
NED
FAM
NED
NED
FAM
PET
VIC
VIC
FAM
VIC
PET
NED
FAM
VIC
PET
NED
VIC
PET
VIC
FAM
NL c
NL c
NL c
FAM
VIC
FAM
NL c
NL c
FAM
NL c

D9Mit182
D9Mit53
D9Mit184
D9Mit20
D9Mit214
D9Mit215
D9Mit281
D9Mit120
D9Mit52 a
D10Mit2 *a
D10Mit31 a
D10Mit266 a
D11Mit21 a
D11Mit67 a
D11Mit48 a
D11Mit184
D12Mit109 a
D12Mit156 a
D12Mit30
D12Mit133 a
D13Mit132 a
D13Mit13 a
D13Mit51 a
D14Mit1 a
D14Mit233 a
D14Mit225 a
D15Mit12
D15Mit13 a
D15Mit85
D15Mit171 a
D15Mit42 a
D16Mit129 *a
D16Mit139 a
D16Mit48
D16Mit106 a
D17Mit163 a
D17Mit138
D17Mit53 a
D17Mit93 a
D18Mit19
D18Mit12 a
D18Mit91 a
D18Mit40
D18Mit187 a
D18Mit25
D19Mit78 a
D19Mit14 a
D19Mit103 a
DXMit55 a
DXMit25 a
DXMit130
DXMit121 a

Position
129S6/SvEv
NOG C57BL/6(cM) b			
(CCE ES)
55
57
60
61
62
63
68
69
72
16
36
62
20
57
77
78
19
34
46
56
4
35
59
3
19.5
42.5
4.7
6.7
16.4
54.5
59.2
3.4
43.1
43.3
71.5
3
24.2
38.5
44.5
2
17
29
37
47
57
5
15
52
1.4
27.8
55
67

115
198
125
95
135
116
106
126
173
133
146
82
147
133
123
ND
124
184
103
96
157
145
139
101
182
94
143
138
194
141
178
161
150
155
132
126
ND
122
141
150
122
135
135
106
117
130
150
117
121
158
161
130

99
206
132
104
137
116
111
146
171
126
148
90
158
131
129
149
116
176
103
111
150
151
137
104
194
111
143
133
194
132
182
180
144
155
142
130
138
128
154
150
110
137
137
110
117
126
153
115
129
168
161
147

104
198
127
114
108
122
106
146
171
133
151
84
147
122
123
160
124
171
113
111
157
145
130
98
179
102
151
116
199
141
159
167
170
159
132
124
129
122
141
133
110
137
137
110
109
128
146
123
127
158
136
147

Dye
NL c
FAM
FAM
FAM
FAM
NED
FAM
NL c
VIC
NED
NED
PET
VIC
VIC
NED
NED
VIC
FAM
PET
FAM
NED
PET
FAM
PET
VIC
FAM
FAM
NED
FAM
FAM
VIC
PET
VIC
VIC
PET
VIC
FAM
VIC
NED
FAM
FAM
PET
FAM
FAM
NED
FAM
PET
NED
NED
VIC
VIC
VIC

: Informative microsatellite markers for distinguishing between the NOG and C57BL/6 strains; b: according to the Mouse Genome Database (MGD; http://www.informatics.jax.org); *: Primers were changed from the original sequence on the NCBI UniSTS database toredesigned sequences in order to change the size of the PCR product as follows; D3Mit149: F 5’-TTCCATACAAACAAAAGCAACG-3’,
R 5’-CTATATAGCTGTAAATGTAAAGTGTATGTC-3’, D8Mit155: F 5’-TTGGACAGGGAAAATTCTGC-3’, R 5’-GAAAATGTGACACCATTTGAGGAC-3’, D10Mit2: F 5’-GTTCATTTGAGGCACAAGCA-3’, R 5’-TTTGAGCTGCTCACAACCC-3’, and D16Mit129: F
5’-ATGAGCAGTCTGCAGACCTT-3’, R 5’-GAGACTGAGAAAGGGGATGC-3’; c: non-labeled primer pairs; ND: not detected.
a474

H. Suemizu, ET AL.
Table 2. Comparison of the methods used to generate NOG-EGFP mice
Generation number a
Expected(Number examined b)
		PF1N2 (13)N3 (15)
N4 (31)N5 (4)

050 (61/122)
75 (91.5/122)
87.5 (106.8/122)
93.8 (114.4/122)
96.9 (118.2/122)% Recipient genome (R/total) c
MAX–
50 (61/122)
81.1 (99/122)
95.9 (117/122) *98.4 (120/122) **99.2 (121/122)

ObservedMIN

AVG ± SD––
66.4 (81/122)
88.5 (108/122)
95.9 (117/122)
98.4 (120/122)––
73.1 ± 4.1
91.3 ± 2.4
96.9 ± 0.8
98.8 ± 0.5

The generation count begins at strain P (parental) defined as 100% original background, 0% recipient background.
The F1 generation contains the offspring from the intercross between P (100% original) × pure recipient NOG
strains (100% recipient). F1 animals have 50% of the recipient genome. b The numbers of transgenic male mice
whose genetic backgrounds were examined. c R: the number of homozygotes for the allele of the recipient strain;
total: total number of alleles. *P=0.015 and **P=0.095 (Fisher’s exact probability test).
aputer simulations of MASP-based congenic strain construction strategies using high-density (10 cM apart) andlow-density (25 cM apart) marker spacing, and screeninga mean of 40 and 16 progeny per generation revealed
that screening 16 progeny per generation with a lowdensity marker was the most cost-effective strategy. Our
MASP strategy is similar to the computer-simulated mostcost-effective strategy because 61 informative microsatellite markers were arranged throughout the mouse genome spaced at an average distance of 26.5 cM (lowdensity), and we screened 13, 15, and 31 progeny at N 2,
N3, and N4, respectively. However, our MASP strategy
included some gaps of over 30 cM, with a largest gap of
51.7 cM. One major problem is that a larger gap might
not be able to detect a double crossing-over occurringin meiosis. Hameister et al. examined the frequency of
double crossing-over in a 55.4-cM region between chromosome 15A2 and 15F2-3 in 151 mice and found only
one animal with a double crossing-over [2]. In reality,
double crossing-over does not occur at a high frequency,
even in a larger gap extending over 50 cM. Therefore,
detecting double crossing-over using adjacent markers
might be possible. Another problem is the inability todetect a small segment of the donor genome. Because
screening all gaps over 30 cM is unrealistic, we screened
one large gap (43 cM) extending between D9Mit97 andD9Mit52 in more detail. We used extremely high-density markers (2.4 cM marker spacing) consisting of 17
informative markers in 12 and 14 progeny at N 2 and N3,
respectively. No animal with a double crossing-over was

found in 12 animals in the N2 generation, while a small
contaminating segment of the donor genome was detected between markers D9Mit207 and D9Mit107 inprogeny #E109 of the N3 generation (Fig. 1). Considering an undetected contaminating donor genome, Wakeland et al. recommended performing one or two additional backcrosses when all of the markers become
recipient-derived. This additional backcross reduces thelevel of undetected contamination in these strains to theequivalent of that at N10 or N11 using the traditional protocol [12]. When transferring a transgene or modified
gene into the NOG or C57BL/6 strain from C57BL/6 or
NOG and 129S6/SvEv backgrounds, our selected 61
markers serve as genetic quality standards for generating
a congenic strain using MASP. The final backcross could
be completed because the NOG-EGFP mouse (N5) met
the genetic quality standards after backcrossing fourtimes. However, we persisted with backcrossing during
the production of congenic progeny derived from the N5generation for xenotransplantation studies. This additional backcross might confer some benefit through thefurther elimination of residual donor genomes.
To examine the phenotype of the NOG-EGFP mice,the male mouse closest to the NOG strain with EGFP
transgene in the N5 generation was mated with female
NOG mice to obtain many NOG-EGFP mice (N6) for
xenotransplantation studies. NOD/Shi-scid mice (male,
10 weeks old), NOG mice (female, 7 weeks old), andNOG-EGFP mice (female, 12 weeks old) were irradiated with 2.4 Gy of X-rays 24 h before transplanting 5

Generation of NOG-EGFP mice

475

Fig. 1. Detailed screening between D9Mit97 and D9Mit52 using extremely high-density markers (2.4 cM marker spacing).
The screening is depicted schematically using genotyping data in which each box indicates one informative microsatellite marker primer pair. The yellow boxes represent those PCR products that are identified as NOG homozygotes based on size. The red boxes represent C57BL/6 and NOG heterozygotes. The asterisks indicate the “best”
males that were selected as the parents of the next backcross generation.

× 104 human CD34+ cells (Lonza Walkersville, Inc.).
The engraftment of donor cells was monitored every 4
weeks by detecting the human cells expressing the leukocyte common Ag CD45 (Immunotech, Marseille,
France) using a MoFlo flow cytometer (Dako, Glostrup,
Denmark) and Summit software. Representative flow
cytometric analysis of peripheral blood of mice that underwent transplantation (after 12 weeks) is shown in Fig.
2a. When using NOG-EGFP mice as recipients, we
could easily differentiate donor human cells from recipient mouse cells with the fluorescent signals of the
EGFP, although the cells were not stained with any cellsurface marker for the mouse species. Twelve weeks
after transplantation, significantly more growth of humancells was observed in both NOG mice and NOG-EGFP
mice compared to NOD/Shi-scid mice (*P<0.01;**P<0.001; Fig. 2b), but there was no significant difference between NOG mice and NOG-EGFP mice. These
results suggest that the NOG-EGFP mice, which weregenerated by the congenic method with MASP, acquired
the immunological properties of the NOG strain.
In this experiment, we did not use X-chromosomederived microsatellite markers as informative markers
to select a parent for the next generation because the

X-chromosome is always derived from the recipient
NOG strain in male transgenic mice. However, wechecked the X-chromosome with informative markers
to distinguish between the NOG and 129S6/SvEv strains(the source of the CCE ES cells) because of the confirmation of an X-chromosome linked IL2Rgnull mutation (ChrX, 38 cM) derived from the recipient genome. However, the nearest marker located at 32.1 cM was not informative, and the other three markers on the X-chromosome were shown to be homozygous for recipientderived alleles.
Nakanishi et al. analyzed transgene integration sites
in more than 100 EGFP transgenic mouse lines rigorously using fluorescent in situ hybridization and determined that the EGFP transgene is located on the D1
region of chromosome 14 in the 131 line (C57BL/6-Tg(Act-EGFP) C14-Y01-FM1310sb) [6]. The microsatellite markers that did not become NOG markers in the N4
generation were the markers D14Mit233 and D14Mit225,
both of which are on chromosome 14. Fortunately, we
could change the genetic background around theD14Mit225 region from C57BL/6 to the NOG strain in
the N5 generation, but the genetic background aroundthe D14Mit233 region remained C57BL/6. D14Mit233

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H. Suemizu, ET AL.

Fig. 2. High engraftment efficiency of human cells in NOG-EGFP mice. (a) Representative flow cytometric data for peripheral blood obtained from NOG-EGFP micethat underwent human cord blood CD34+ cell transplantation (12 weeks after
transplantation). (b) Comparison of the engraftment levels of human cells in
NOD/Shi-scid, NOG, and NOG-EGFP mice. At the indicated times following
transplantation of 5 × 104 CD34+ cells, human CD45+ cells in mouse peripheral
blood were assayed by flow cytometry (n=3 each). **P<0.01, ***P<0.001.

and D14Mit225 are located in the B and D1–D3 regions,
respectively. These markers are located extremely close
to the site of EGFP transgene integration in the C5BL/6TgEGFP line 131. The D14Mit233 marker seemed to
be closer to the integrated transgene than the D14Mit225
marker because six recombinants at the D14Mit225 locus
were observed in the 63 male Tg EGFP mice, while norecombinant was observed for the D14Mit233 marker.
The distance from the D14Mit225 marker to the integrated EGFP transgene can be calculated as roughly 9.5cM based on the recombination frequency [10]. Basedon statistical modeling, the traditional 12 backcross generation (N12) protocol will produce a congenic strain inwhich more than 99% of the genome is unlinked to thetarget gene carried in a donor-derived genome segmentwith an average length of about 20 cM [1]. Therefore,
the D14Mit233 marker genetically linked to the transgene integration site is physically close enough to thetransgene that it will not segregate independently duringmeiosis.
Acknowledgment(s)We thank Noriko Omi and Chika Kito of CIEA for
their technical assistance and Dr. Masaru Okabe (Osaka
University) for providing the green mice.

Generation of NOG-EGFP mice

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