Disruption of the gene encoding 3b-hydroxysterol
D
14
-reductase (Tm7sf2) in mice does not impair
cholesterol biosynthesis
Anna M. Bennati
1
, Gianluca Schiavoni
1
, Sebastian Franken
2
, Danilo Piobbico
3
, Maria A. Della
Fazia
3
, Donatella Caruso
4
, Emma De Fabiani
4
, Laura Benedetti
5
, Maria G. Cusella De Angelis
5
,
Volkmar Gieselmann
2
, Giuseppe Servillo
3
, Tommaso Beccari
1

Fax: +39 075 585 7428
Tel: +39 075 585 7426
E-mail: [email protected]
(Received 24 May 2008, accepted 11
August 2008)
doi:10.1111/j.1742-4658.2008.06637.x
Tm7sf2 gene encodes 3b-hydroxysterol D
14
-reductase (C14SR, DHCR14),
an endoplasmic reticulum enzyme acting on D
14
-unsaturated sterol interme-
diates during the conversion of lanosterol to cholesterol. The C-terminal
domain of lamin B receptor, a protein of the inner nuclear membrane
mainly involved in heterochromatin organization, also possesses sterol
D
14
-reductase activity. The subcellular localization suggests a primary role
of C14SR in cholesterol biosynthesis. To investigate the role of C14SR and
lamin B receptor as 3b-hydroxysterol D
14
-reductases, Tm7sf2 knockout
mice were generated and their biochemical characterization was performed.
No Tm7sf2 mRNA was detected in the liver of knockout mice. Neither
C14SR protein nor 3b-hydroxysterol D
14
-reductase activity were detectable
in liver microsomes of Tm7sf2
() ⁄ ))
mice, confirming the effectiveness of

8,14
, 4,4-dimethyl-5a-cholesta-8(9),14-dien-3b-ol; ER, endoplasmic reticulum; HEM, Hydrops-
Ectopic calcification-Moth-eaten skeletal dysplasia; LBR, lamin B receptor.
5034 FEBS Journal 275 (2008) 5034–5047 ª 2008 The Authors Journal compilation ª 2008 FEBS
[1,2]. A second protein of the inner nuclear membrane,
the lamin B receptor (LBR), catalyzes the 3b-hydroxys-
terol D
14
-reductase reaction, as demonstrated by its
ability to complement C14SR-deficient yeast strains
[3,4] and by enzymatic assay of the protein overexpres-
sed in transfected COS-1 cells [5]. Recently, the mouse
gene encoding 3b-hydroxysterol D
14
-reductase has been
termed Dhcr14 [6]; in this study the former gene name
Tm7sf2 will be used. Tm7sf2 is located on chromosome
19A.
The expression of cholesterol biosynthesis genes is
regulated by cell sterol levels through the action of the
transcription factor SREBP-2 [7,8]. In HepG2 hepa-
toma cells, sterol starvation results in induction of the
TM7SF2 gene, C14SR protein and 3b-hydroxysterol
D
14
-reductase activity. In addition, human TM7SF2
promoter is regulated by SREBP-2 [5]. Therefore, the
adaptability of TM7SF2 gene to the needs of choles-
terol biosynthesis appears well established. On the con-
trary, LBR gene expression is not responsive to sterol

or defective for Dhcr14 ⁄ Tm7sf2 and heterozygous for
Lbr (Lbr
(+ ⁄ ))
:Dhcr14
(D4-7 ⁄ D4-7)
) [6]. The paper states
that HEM dysplasia is a laminopathy not caused by
3b-hydroxysterol D
14
-reductase deficiency. Mutants
have distinct physical and biochemical phenotypes, but
no sterol abnormalities were detected in liver, whereas
marked elevations of D
14
-sterols were seen in brain of
Lbr
(+ ⁄ ))
:Dhcr14
(D4-7 ⁄ D4-7)
mice.
Here, we describe the generation of Tm7sf2
() ⁄ ))
mice and their biochemical characterization. Determi-
nation of Tm7sf2 and Lbr mRNA expression in differ-
ent mouse tissues, expression of C14SR and LBR
proteins in liver and a measure of their 3b-hydroxys-
terol D
14
-reductase activity are reported. Despite the
lack of C14SR, Tm7sf2

3 (55 bp) 3 (274 bp) CCTATTAATG gtgactgggg–––tgtggttcag GCTTCCAGGC
4 (195 bp) 4 (84 bp) GGAAACTCAG gtgagaaggg–––ttgttcccag GAAATTCCAT
5 (104 bp) 5 (2112 bp) CATTGGCTGG gtatgctgac–––acttctttag GTTTTCATTA
6 (120 bp) 6 (88 bp) CTGGTATGAG gtgagactgg–––gttcctgcag GAGTCTGTCC
7 (169 bp) 7 (214 bp) CTCCTTAAGG gtcagtagga–––cttccctcag TTATTGGTTA
8 (81 bp) 8 (80 bp) AGCGTGGCTG gtaagctggg–––gtatttctag GTCTTGAGAC
9 (123 bp) 9 (250 bp) TTGCCCTGTG gtgagtgggt–––ttccctccag GGCTATCCCA
10 (253 bp) CTATCCCATC–––
A. M. Bennati et al. Tm7sf2 knockout mice
FEBS Journal 275 (2008) 5034–5047 ª 2008 The Authors Journal compilation ª 2008 FEBS 5035
GT–AG rule. The transcription initiation site, deter-
mined by RACE, was located at )91 bp upstream the
ATG start codon. A polyadenylation signal
(AATAAA) is present 49 bp downstream of the stop
codon. The genomic sequence has been submitted to
GenBank under accession number EU672836.
Tm7sf2 and Lbr expression in mouse tissues
Tm7sf2 and Lbr relative mRNA expression was mea-
sured in adrenal, brain, heart, kidney, liver, lung,
ovary and testis of 8-week-old mice using qRT-PCR.
The highest Tm7sf2 mRNA abundance was found in
liver, followed by ovary, testis, kidney and brain
(Fig. 1A). Testis and lung showed the highest Lbr gene
expression, followed by heart, ovary, kidney and liver
(Fig. 1B).
Tm7sf2 versus Lbr expression was determined by
using Lbr as the internal calibrator for each tissue.
Table 2 shows comparable expression of the two genes
in ovary, kidney and adrenal gland. Compared with
Lbr,  8- and 2.5-fold higher Tm7sf2 expression was

ney, adrenal and brain did not reveal differences
between control and mutant mice (data not shown).
Followed over a 3-month period, Tm7sf2
() ⁄ ))
mice
grow at the same rate as littermate control mice.
Groups of control and Tm7sf2
() ⁄ ))
female weighed at
14 months of age were 27.7 ± 1.9 g (n = 9) and
30.9 ± 1.9 g (n = 7), respectively. No apparent age-
dependent problems were observed in females or males
over a 14-month period. These results confirm previ-
ously reported data [6].
0.0
0.2
0.4
0.6
0.8
1.0
1.2
A
B
Tm7sf2 relative mRNA expression
Adrenal
Brain
Heart
Kidney
Liver
Lung

and reverse primers that amplify the entire cDNA.
Figure 3A shows that the cDNA is absent in
Tm7sf2
() ⁄ ))
mice, whereas the expected 1.3 kb frag-
ment is obtained in control mice. Although no quanti-
tative PCR was performed, the cDNA was about half
of the control in heterozygous mice. Gapdh was ampli-
fied in parallel as housekeeping gene.
Western blot analysis of microsomes prepared from
liver was performed using anti-(bovine C14SR) serum,
which cross-reacts with the mouse protein. Figure 3B
shows that C14SR protein is absent in Tm7sf2
() ⁄ ))
mice, whereas the band intensity is about half of
control (0.55 ± 0.09) in heterozygous mice, thus con-
firming the results obtained with the cDNA.
To investigate whether disruption of the Tm7sf2
gene modifies Lbr mRNA expression, qRT-PCR was
performed in tissues of 8-week-old wild-type and
Tm7sf2
() ⁄ ))
mice, using the wild-type as internal cali-
brator for each tissue. No significant differences of Lbr
mRNA expression in Tm7sf2
() ⁄ ))
mice, compared
with that of wild-type mice, were found in the exam-
ined tissues (adrenal, brain, heart, kidney, liver, lung,
ovary and testis) (data not shown).

the disrupted allele. (D) Southern blot analy-
sis of mouse tail DNA isolated from the
progeny of a mating between heterozygous
parents. DNAs were digested with EcoRI
and hybridized with the 3¢-probe indicated in
(B).
Table 2. Tm7sf2 and Lbr expression in mouse tissues. Total RNA
was extracted from tissues of 8-week-old mice and retrotranscribed
as reported in Experimental procedures. Tm7sf2 and Lbr mRNA
expression was measured by qRT-PCR using the specific primers
(see Experimental procedures) and Lbr as internal calibrator for
each tissue. Hprt was used as the reference gene for sample nor-
malization. Data are mean ± SD of two experiments performed in
triplicate.
Tissue Tm7sf2 ⁄ Lbr ratio
Adrenal 0.68 ± 0.06
Brain 2.40 ± 0.51
Heart 0.09 ± 0.03
Kidney 0.90 ± 0.09
Liver 7.95 ± 0.71
Lung 0.06 ± 0.01
Ovary 1.06 ± 0.13
Testis 0.19 ± 0.05
A. M. Bennati et al. Tm7sf2 knockout mice
FEBS Journal 275 (2008) 5034–5047 ª 2008 The Authors Journal compilation ª 2008 FEBS 5037
3b-Hydroxysterol D
14
-reductase activity
3b-Hydroxysterol D
14

14
-reductase reaction in liver from wild-
type mice can be evaluated. On the basis of the
amount of incubated proteins (see legend in Fig. 4) in
the experimental conditions used, C14SR-specific activ-
ity was approximately eightfold higher than that of
LBR. Because 4.4 and 1.5 mg proteinÆg
)1
of fresh tis-
sue were recovered as microsomes and nuclei, respec-
tively, C14SR enzymatic activity is >20-fold higher
than LBR enzymatic activity. This result is in accor-
dance with the high Tm7sf2 mRNA expression in liver,
compared with Lbr (Table 2).
Sterol determinations
Cholesterol concentration in liver microsomal mem-
branes of 6-week-old mice was measured both by
GC-MS analysis and by densitometry analysis of mem-
brane lipids separated by TLC. Despite the lack of
C14SR activity in microsomes, normal cholesterol
levels were found in these membranes (Table 3). No
differences were found between male and female mice.
Cholesterol biosynthetic precursors, including
C29D
8,14
, were not detectable by GC-MS analysis
of microsomal sterols, indicating that C29D
8,14
inter-
mediate was not accumulated in Tm7sf2

1.0
1.5
2.0
2.5
Sterol/cholestane (peak area ratio)
Nuclei
t
0
+/+ +/– –/– +/+ –/–
Microsomes
Fig. 4. 3b-Hydroxysterol D
14
-reductase activity. Microsomes
(0.25 mg protein) and intact nuclei (0.5 mg protein) prepared from
liver of wild-type, heterozygous and Tm7sf2
() ⁄ ))
mice were
assayed for 3b-hydroxysterol D
14
-reductase activity by incubation
for 30 min with C27D
8,14
in the conditions described in Experimen-
tal procedures. Enzymatic activity was evaluated on the basis of
the decrease of peak area ratio between m ⁄ z 426 and m ⁄ z 372
ions (C27D
8,14
⁄ 5a-cholestane, filled columns) and the increase of
peak area ratio between m ⁄ z 428 and m ⁄ z 372 ions (C27D
8

SD < 0.2 were selected in the comparison knock-
out ⁄ wild-type mice. Volcano Plot analysis of this tran-
script list was performed to identify transcripts with a
defined minimal fold change and statistically significant
P-value for a t-test of differences between samples. By
selecting a fold change > 1.5 and a P-value < 0.01, 66
transcripts were identified as increased (Table 4) and 41
as decreased (Table 5) in livers of Tm7sf2
() ⁄ ))
mice.
Table 4 shows that several transcripts of oxidoreduc-
tases are increased, including members of cyt p450
families. Glutathione S-transferase, involved in xenobi-
otic metabolism, is also increased. Some genes involved
in cell proliferation and cell-cycle progression show
decreased transcripts (Table 5). qRT-PCR analysis was
applied to some of the genes that show the highest
up- or down expression comparing knockout to the
wild-type mice. Although with different fold changes,
the results obtained in the microarray experiment were
confirmed (Tables 4 and 5).
The complete panel of genes upstream and down-
stream Tm7sf2 in the cholesterol biosynthetic pathway
and Lbr were analysed carefully using less stringent
parameters (P-value for the t-test < 0.05). Neverthe-
less, no difference in their expression was found in the
liver of Tm7sf2
() ⁄ ))
mice.
Discussion

sion in liver microsomes of Tm7sf2
(+ ⁄ ))
and
Tm7sf2
() ⁄ ))
mice, respectively. In accordance with
these data, no 3b-hydroxysterol D
14
-reductase activity
is detectable in liver microsomes of Tm7sf2
() ⁄ ))
mice.
Despite the lack of 3b-hydroxysterol D
14
-reductase
activity of C14SR, normal cholesterol biosynthesis
occurs in Tm7sf2
() ⁄ ))
mice. Indeed, the level of
Table 3. Cholesterol in plasma and liver microsomal membranes. Total plasma cholesterol was measured using a commercial kit. For micro-
somal membrane cholesterol determination, samples were saponified and cholesterol was measured by GC-MS and by densitometric analy-
sis of lipids separated by TLC and stained as described in Experimental procedures.
Plasma cholesterol (mgÆdL
)1
)
Microsomal membrane cholesterol (nmolÆmg
)1
protein)
Tm7sf2
(+ ⁄ +)

1420603_s_at NM_009016, NM_009017,
NM_009018, NM_020030,
NM_198193, XM_001006217
Raet1a, Raet1b,
Raet1c, Raet1d,
Raet1e
retinoic acid early transcript
1alpha, beta, gamma, delta, 1E
3.18
1449347_a_at NM_001081642, NM_021365,
NM_183094, XM_001471704,
XM_001471888, XM_001475552,
XM_001487778, XM_978371,
XR_035676, XR_035679
LOC100044048,
LOC100044049,
LOC100046087,
Xlr4a, Xlr4b,
Xlr4c, Xlr4e
X-linked lymphocyte-regulated
4A, 4B, 4C, 4E
3.02
1444438_at XM_356089, XM_904518 Cib3 calcium and integrin binding
family member 3
2.98 ⁄ 1.66
a
1424853_s_at NM_010011, NM_201640,
XM_001471913
Cyp4a10, Cyp4a31,
LOC100044218

Gsta1, Gsta2,
LOC100042295
glutathione S-transferase, alpha 1
(Ya); glutathione S-transferase,
alpha 2 (Yc2)
2.06
1438194_at – 2900019G14Rik RIKEN cDNA 2900019G14 gene 2.06
1456973_at – – – 2.05
1452501_at NM_010002 Cyp2c38 cytochrome P450, family 2,
subfamily c, polypeptide 38
1.98
1422903_at NM_010745 Ly86 lymphocyte antigen 86 1.93
1415932_x_at NM_015731 Atp9a ATPase, class II, type 9A 1.92
1418213_at NM_033373 Krt23 keratin 23 1.92
1444706_at – Nav2 neuron navigator 2 1.91
1423627_at NM_008706 Nqo1 NAD(P)H dehydrogenase, quinone 1 1.88
1450505_a_at NM_001034851, NM_025459 1810015C04Rik RIKEN cDNA 1810015C04 gene 1.85
1450648_s_at NM_207105 H2-Ab1 histocompatibility 2, class II
antigen A, beta1
1.84
1417900_a_at NM_013703 Vldlr very low density lipoprotein receptor 1.83
1455316_x_at XM_915804 ENSMUSG00000073624 predicted gene, ENSMUSG00000073624 1.81
1458585_at – – – 1.80
1447643_x_at NM_011415 Snai2 snail homolog 2 (Drosophila) 1.78
Tm7sf2 knockout mice A. M. Bennati et al.
5040 FEBS Journal 275 (2008) 5034–5047 ª 2008 The Authors Journal compilation ª 2008 FEBS
cholesterol in microsomes from liver, as well as plasma
cholesterol, were comparable between wild-type and
Tm7sf2
() ⁄ ))

1427604_a_at NM_015731 Atp9a ATPase, class II, type 9A 1.78
1439293_at NM_153584 BC031353 cDNA sequence BC031353 1.76
1419430_at NM_007811 Cyp26a1 cytochrome P450, family 26, subfamily a,
polypeptide 1
1.75
1450884_at NM_007643 Cd36 CD36 antigen 1.74
1420879_a_at NM_018753 Ywhab tyrosine 3-monooxygenase ⁄ tryptophan
5-monooxygenase activation protein,
beta polypeptide
1.73
1429831_at NM_031376 Pik3ap1 phosphoinositide-3-kinase adaptor protein 1 1.72
1418710_at NM_007652 Cd59a CD59a antigen 1.71
1448978_at NM_019867 Ngef neuronal guanine nucleotide exchange factor 1.68
1446731_at – A730016A17 Fanconi anemia, complementation group F 1.68
1417025_at NM_010382 H2-Eb1 histocompatibility 2, class II antigen E beta 1.68
1422975_at NM_008604 Mme membrane metallo endopeptidase 1.63
AFFX-r2-Bs-
thr-M_s_at
– – – 1.63
1417629_at NM_011172 Prodh proline dehydrogenase 1.63
1417017_at NM_007809 Cyp17a1 cytochrome P450, family 17, subfamily
a, polypeptide 1
1.63
AFFX-ThrX-M_at – – – 1.62
1431916_at NM_001012306 Hsd3b3 hydroxy-delta-5-steroid dehydrogenase,
3 beta- and steroid delta-isomerase 3
1.62
1417828_at NM_007474 Aqp8 aquaporin 8 1.61
1448595_a_at NM_009052 Bex1 brain expressed gene 1 1.60
1428083_at NR_003513, XR_035481,

probeset
identification GenBank Gene symbol Gene name Fold change
1444297_at NR_002861, XM_001471933 LOC100044164,
Serpina4-ps1
serine (or cysteine) peptidase inhibitor,
clade A, member 4, pseudogene 1
)5.26
1444296_a_at NR_002861, XM_001471933 LOC100044164
Serpina4-ps1
serine (or cysteine) peptidase inhibitor,
clade A, member 4, pseudogene 1
)4.05
1448092_x_at NR_002861, XM_001471933 LOC100044164
Serpina4-ps1
serine (or cysteine) peptidase inhibitor,
clade A, member 4, pseudogene 1
)4.02
1427797_s_at NM_007799 Ctse cathepsin E )3.92-
1416664_at NM_023223 Cdc20
b
cell division cycle 20 homolog (S. cerevisiae) )3.60 ⁄ )10.8
a
1424638_at NM_007669 Cdkn1a
b
cyclin-dependent kinase inhibitor 1A (P21) )3.38 ⁄ )8.9
a
1417764_at NM_025965, XM_911969 LOC636537, Ssr1 signal sequence receptor, alpha )3.30
1420451_at NM_021370 Accn5 amiloride-sensitive cation channel 5,
intestinal
)3.23 ⁄ )4.1

predicted gene, EG434175
)2.13
1422001_at NM_010565 Inhbc inhibin beta-C )2.10
1423397_at NM_133894 Ugt2b38 UDP glucuronosyltransferase 2 family,
polypeptide B38
)2.08
1419669_at NM_011178 Prtn3 proteinase 3 )1.98
1417370_at NM_011575 Tff3 trefoil factor 3, intestinal )1.96
1424695_at NM_025912 2010011I20Rik RIKEN cDNA 2010011I20 gene )1.93
1442051_at NM_013549 Hist2h2aa1 histone cluster 2, H2aa1 )1.92
1437073_x_at – AV025504 expressed sequence AV025504 )1.89
1450440_at NM_010279 Gfra1 glial cell line derived neurotrophic
factor family receptor alpha 1
)1.84
1424118_a_at NM_025565 Spc25
b
SPC25, NDC80 kinetochore complex
component, homolog (S. cerevisiae)
)1.83
1416299_at NM_011369 Shcbp1
b
Shc SH2-domain binding protein 1 )1.82
1433955_at NM_145125 Brwd1
b
bromodomain and WD repeat domain
containing 1
)1.77
1419319_at NM_011316 Saa4 serum amyloid A 4 )1.77
1448314_at NM_007659 Cdc2a
b

and we did not find significant differences between wild-
type and Tm7sf2
() ⁄ ))
mice. In the liver, the result
obtained by qRT-PCR was further confirmed by the
Affymetrix oligonucleotide array hybridization experi-
ment. In addition, the expression of LBR protein in
nuclei from liver and its enzymatic activity were compa-
rable in wild-type and Tm7sf2
() ⁄ ))
mice. These results
indicate that, at least in adult Tm7sf2
() ⁄ ))
mice, LBR
can account for normal cholesterol biosynthesis without
increasing its expression. The discrepancy in Lbr gene
expression between 1-day-old Dhcr14
(D4-7 ⁄ D4-7)
and
adult Tm7sf2
() ⁄ ))
mice could be related to higher cho-
lesterol biosynthetic activity during development, com-
pared with adult mice. In vitro, C14SR exhibits higher
cholesterol biosynthetic capacity than LBR. Although it
cannot be excluded that this is due to the experimental
conditions used for enzymatic activity determination
(substrate and ⁄ or cofactors), this result is in accordance
with higher Tm7sf2 gene expression in liver, compared
with Lbr. In vivo, the regulation of Tm7sf2 gene expres-

should occur.
The role of cholesterol and intermediates of its bio-
synthesis in cell growth and division is well known
[28,29]. The stringency of the requirement for choles-
terol during proliferation and cell-cycle progression
was investigated in promyelocytic HL-60 cells by com-
parison with other sterols of the biosynthetic pathway.
In the absence of exogenous cholesterol, accumulation
of intermediate sterols upstream 7-dehydrocholesterol,
including C29D
8,14
, resulted in the inhibition of cell
proliferation and cell cycle arrest in G2 ⁄ M phase [30].
Affymetrix oligonucleotide array analysis showed that
several genes involved in cell proliferation and cell-
cycle progression have decreased expression in the liver
of Tm7sf2
() ⁄ ))
mice. Although no altered phenotype
has been observed in Tm7sf2
() ⁄ ))
mice so far, we
could speculate that an impaired response of liver cells
to proliferative stress is conceivable in these mice.
The evaluation of C14SR and LBR expression and
the determination of their enzymatic activity in the
liver of wild-type and Tm7sf2
() ⁄ ))
mice reinforce the
hypothesis that LBR and C14SR provide enzymatic

tease inhibitor cocktail tablets were from Roche Diagnos-
tics (Milan, Italy). RNAlater RNA Stabilization Reagent,
Qiazol Lysis Reagent, and RNeasy Mini Kit were from
Qiagen (Milan, Italy). QuickChange Site-Directed Muta-
genesis Kit, AffinityScript Multiple Temperature Reverse
Transcriptase, and Brilliant
Ò
SYBR
Ò
Green QPCR Master
Mix were purchased from Stratagene (La Jolla, CA, USA).
RiboLock RNase inhibitor, random hexamer primers, Taq
DNA polymerase, and restriction enzymes were from
Fermentas (St Leon-Rot, Germany). 5a-Cholesta-8,14-dien-
3b-ol was synthesized as previously described [2].
A. M. Bennati et al. Tm7sf2 knockout mice
FEBS Journal 275 (2008) 5034–5047 ª 2008 The Authors Journal compilation ª 2008 FEBS 5043
Identification of mouse Tm7sf2 gene
Mouse Tm7sf2 mRNA was identified by NCBI database
comparison with the human mRNA (accession no.
AF096304). The mouse cDNA was synthesized by RT-PCR
using liver RNA as template and the following primers: for-
ward 5¢-ATGTCGACGATCATGACTTCTCGTGAGG-3¢
and reverse 5¢-ATGTCGACTTCAACCTCTTAGGTG
GACC-3¢ (the SalI restriction site introduced for subclon-
ing in pBlueScript vector is given in italics). The sequenced
1285 bp cDNA (accession no. AF480070) encodes a puta-
tive protein 86% identical to human C14SR [1,2].
The cDNA was used to screen a 129 ⁄ SvJ mouse genomic
library in Lambda FIX II vector (Stratagene). Five library

Targeted embryonic stem cell clone E-53 was injected
into C57 ⁄ B6 blastocysts to generate chimeric mice. Chime-
ric males were mated with C57 ⁄ B6 females and progenies
were analysed for germline transmission of the mutated
allele. Unless otherwise specified, the experiments were per-
formed with 129 ⁄ Sv-C57 ⁄ B6 hybrid descendants (F1) of
these animals. Backcross into C57 ⁄ B6 was also carried out
for four generations to obtain mice with > 90% C57 ⁄ B6
genetic background. All experiments involving animals were
conducted according to protocols approved by the
Bioethics Committee of University of Perugia.
Mice were genotyped for the introduced Tm7sf2 mutated
gene by PCR analysis of tail genomic DNA using three spe-
cific primers (a, 5¢-AAGGCTTTGGTAGCTCCTGCCT-3¢;
b, 5¢-TGAGGCCAGGTCTCAGCTCAC-3¢; neo, 5¢-GCT
ATCAGGACATAGCGTTGGC-3
¢; see Fig. 2B). PCR
cycling conditions were: 2 min denaturation at 95 °C fol-
lowed by 35 cycles of 30 s at 95 °C, 45 s at 65 °C, 30 s at
72 °C, and a final extension of 5 min at 72 °C. Mouse
genotype was confirmed by Southern blotting analysis.
5¢ RACE
The transcription start site of the mouse Tm7sf2 gene was
determined by 5¢-RACE [34]. Mouse liver RNA was reverse
transcribed using the primer 5¢-AGGAGCTACCAAAGC
CTTCG-3¢ (nucleotides +468 to +449 from the ATG start
codon). A homopolymeric A-tail was then added to the
3¢-end using terminal transferase and dATP. The tailing
product was purified and amplified using the nested reverse
primer 5¢-CGACTCTTGTCCTTCAGTTCC-3¢ (nucleotides

Tm7sf2 knockout mice A. M. Bennati et al.
5044 FEBS Journal 275 (2008) 5034–5047 ª 2008 The Authors Journal compilation ª 2008 FEBS
difluoride) membranes after protein transfer. Labelled pro-
teins were detected by the enhanced chemiluminescence
assay, images were acquired using the VersaDoc Imaging
System, and signals were quantified using quantity one
software (Bio-Rad, Milan, Italy).
3b-Hydroxysterol D
14
-reductase activity
Determination of 3b-hydroxysterol D
14
-reductase activity
was performed by incubating the enzyme source for 30 min
at 37 °C with 5a-cholesta-8,14-dien-3b-ol (C27D
8,14
) [39]. To
measure the C27D
8,14
substrate and the 5a-cholesta-8-en-3b-
ol product (C27D
8
), sterols were purified and analysed by
GC-MS using 5a-cholestane internal standard as previously
described [2]. 3b-Hydroxysterol D
14
-reductase activity was
evaluated on the basis of peak area ratios between m ⁄ z 426
and m ⁄ z 372 ions (C27D
8,14

Reagent (Qiagen) were homogenized using Qiazol Lysis
Reagent (Qiagen). Total RNA was extracted according to
manufacture’s instructions and then subjected to clean up
on mini columns (RNeasy Mini Kit, Qiagen). RNA was
reverse transcribed using AffinityScript Multiple Tempera-
ture Reverse Transcriptase and random hexamer primers.
qRT-PCR amplifications were performed using Mx3000PÔ
Real-Time PCR System with BrilliantÒ SYBRÒ Green
QPCR Master Mix (Stratagene) and ROX as reference dye.
Tm7sf2 specific primers were: forward, 5¢-GCCTCGGTTC
CTTTGACTTC-3¢; reverse, 5¢-CCATTGACCAGCCACAT
AGC-3¢. Lbr specific primers were: forward, 5¢-GTGCTCC
TGAGTGCTTAC-3¢; reverse, 5¢-GCCAATGAAGAAGT
CGTAC-3¢. Housekeeping control was mouse Hprt [42].
Experiments were performed in triplicate and repeated
twice with different RNA preparations. Results were
analysed using mx3000pÔsystem software (Stratagene).
The specificity of the amplified products was assessed by
melting curve.
Affymetrix oligonucleotide array hybridization
and data analysis
Gene expression analysis was performed in liver of
6-week-old wild-type and knockout male mice after the
fourth backcross generation. Total RNA of pooled liver
tissue from three animals was extracted as described
above.
Probe synthesis, hybridization, and data analysis were
performed at the Affymetrix Microarray Unit (Campus
IFOM IEO, Milan, Italy). Biotin-labelled cRNA targets
were synthesized starting from 3 lg of total RNA. Double

GTAAGTGCTGTAGGTAATGAAG-3¢. Onecut1: forward,
5¢-CCTCTATGAATAACCTCTATACC-3¢; reverse, 5¢-TG
CTGGGAGTTGTGAATG-3¢.
A. M. Bennati et al. Tm7sf2 knockout mice
FEBS Journal 275 (2008) 5034–5047 ª 2008 The Authors Journal compilation ª 2008 FEBS 5045
Acknowledgements
The financial support of Telethon–Italy (Grant no.
GGP030102) is gratefully acknowledged. We would
like to thank Aurelio Toia for assistance in GC-MS
analysis of sterols.
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