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 Pdf file on Generation of sp3111 transgenic RNAi
mice via permanent integration of small hairpin RNAs in repopulating
spermatogonial cells in vivo
Weili Shi1, Tingyan Shi2, Zheyu Chen3,
Jufang Lin2, Xiaofeng Jia2, Jian Wang1,2*, and
Huijuan Shi1,2*
1Shanghai 2National Population and Family Planning Committee Key Laboratory of
Contraceptive Drugs & Devices, Shanghai Institute of Planned Parenthood
Research, Shanghai 200032, China
3Department of Neurobiology, *Correspondence address. Tel/Fax: +86-21-64046772; E-mail: [email protected] (H.S.).
[email protected] (J.W.)
The spermatid-specific gene, sp3111, is a new member of the
four-transmembrane gene family. However, its reproductive biological function
remains elusive. The aim of this study was to establish an sp3111-knockdown male
mouse model for further studying the effect of suppression of sp3111 gene expression
on its reproductive functions. Recombinant vectors of pSUPER-sp3111-shRNA,
containing the transcribed functional small hairpin RNA sequence against
different regions of mouse sp3111 mRNA, were constructed and identified. Then, the
linearized recombinant vectors were injected into mature mouse testes and
transfected to spermatogonial cells by electroporation to generate sp3111 transgenic RNAi
mice. These electroporated male mice were mated with wild-type females 30 days
after electroporation and their progenies were examined both by fluorescence
microscopy and PCR. It was found that the suppression efficiency of RNAi on the
testis expression of sp3111 mRNA in vivo was more than 30% and could be transmitted stably to F3
generation, and compared with the wild-type male mice, the mean number of
offspring of the sp3111 RNAi male mice was reduced from 11±1.3 to 6±1.4. In conclusion, the
intra-testicular microinjection technique could also be used to develop the
transgenic RNAi mice, and sp3111 transgenic RNAi mouse model may provide the possibility
to further study the sp3111 gene reproduction function in vivo.
Keywords sp3111 gene; electroporation; transgenic RNAi mice; spermatogonial cell
Received: September 25, 2009 Accepted: November 5, 2009
Introduction
Mammalian
fertilization is a programmed process that includes a series of cellular
interactions terminating with the sperm–oocyte fusion.
Understanding the molecular mechanisms of fertilization contributes not only to
the improvement of clinical treatment for human infertility, but also to the
development of novel targets for contraceptives. Sperm–oocyte fusion is the
culminant step of fertilization; however, its molecular events have not been
completely understood, although it has been well demonstrated that sperm
membrane proteins might play essential roles in this process. Thus,
identification and characterization of novel molecules that are involved in
protein–protein and protein–carbohydrate
interactions between sperm and egg would undoubtedly accelerate a better
insight into the molecular models for fertilization [1,2].
sp3111, a new member of four-transmembrane gene family, is a
spermatid-specific gene (GenBank accession no. NM-000139; 9.8 kb) originally
identified in rat by Shi et al. [3]. The mouse sp3111 cDNA sequence was
subsequently registered in GenBank (GenBank accession no. XM-357051; 3869 bp).
It was found that sp3111 protein was localized in sperm acrosome of rat and the
treatment of rat sperms with polyclonal antibodies against sp3111 protein
before the in vitro fertilization resulted in the abnormal cleavage of
fertilized ova afterward (data not shown). In addition, it has been reported
that some members of another four-transmembrane protein family, tetraspanins,
are expressed in mouse egg, and these egg tetraspanins are involved in the
regulation of sperm–egg fusion by associating with other egg membrane
proteins [4]. Therefore, we speculated that sperm sp3111 might also play an
important role in early embryo development [5]. Transgenic RNAi technology is a
simpler, faster and less expensive approach that knocks down the expression of
target gene in vivo stably. It can be a very effective complementary approach
for knockouts. In the early application of RNAi, siRNAs are commonly used in vitro; they can be readily
transfected into cells, resulting in the transient silence of specific gene
expression. To achieve a more sustained gene-silencing effect, shRNA (small
hairpin RNA) expressed by a vector is preferred. The successful application of
RNAi using the gene-based constructs in mammalian cells led to the idea that
gene silencing might be applied in transgenic mice. Kunath et al. [6] generated
transgenic RNAi mice with Rasa1 knockdown by generating an shRNA vector and introducing
shRNA constructs into the mouse ES cells. Shoji et al. [7] first carried
out in vivo shRNA injection and electroporation of the testis to provide a tool for
assessment of physiological functions of spermatogenic genes in vivo.
In the present study,
we generated transgenic RNAi mice with sp3111 knockdown by
transfecting the inducible shRNA constructs into spermatogonial cells in testis
by in vivo electroporation to observe the effect of suppression of sp3111 gene on male fertility
in mice, with a view to confirm whether or not sp3111 is essential for
fertilization, and to provide a powerful tool for further studies on the
physiological function of sp3111 and its underlying molecular mechanisms in fertilization.
Materials and Methods
Animals
Adult imprinting
control region (ICR) mice aged 4–6 weeks were purchased from SIPPR-BK Laboratory Animal
Company (
Construction of
recombinant pSUPER-sp3111-shRNA vectors
To construct the
expression vector of shRNA that can specifically inhibit the sp3111 gene expression, we
selected three specific sequences (19 bp) according to the sequence of mouse sp3111 mRNA (1335–1353,
1373–1391, and 1370–1388), and then designed three pairs of oligonucleotides
(64 nt). These three pair oligos for hairpin RNA expression that targeted sp3111 gene were chemically
synthesized and annealed. pSUPER vector was linearized by the digestion with BamHI and HindIII (TaKaRa,
Spermatogonia-mediated
gene transfer via electroporation
The recombinant
pSUPER-sp3111-shRNA plasmid was linearized by digestion with PvuI (TaKaRa).
Spermatogonia-mediated gene transfer via electroporation was performed as
described previously [8]. Briefly, ICR male mice of 35 days old were
anesthetized by intraperitoneal injection of pentobarbital sodium. Hair was
removed from the lower abdominal area and the surgical site was cleaned using
75% alcohol, then ventral cutaneous was cut using sterile surgical instruments.
The fat pad associated with the testis was pulled out from the lower side of
the abdominal cavity with the help of curved forceps. Twenty microliters of
linearized pSUPER-sp3111-shRNA constructs (0.5 mg/ml) were injected slowly into the testis at two different
sites using Microliter Syringe (Hamilton,
Generation and
determination of sp3111 transgenic RNAi mice
We refer to the
electroporated males as prefounders. After electroporation, the prefounders
were mated with two wildtype females during days 30–85 and days 115–165,
respectively. Offspring of these mated females were analyzed by fluorescence
microscope (LEICA DFC360 FX; Leica,
Real-time RT-PCR
analysis
Total RNA was prepared
from the testis of 90-day old F1 transgenic lines and wild-type male mice of
90-day old using Trizol (Invitrogen,
Assessment of
male transgenic mice fertility
Both transgenic and
wild-type male mice were successively mated with the same batch of fertile
wide-type female. The offspring number birthed by each of mated females was
documented within 24 h after birth.
Statistical
analysis
The SPSS l1.5 statistical
package was used for data analysis. P < 0.05 was considered statistically
significant. Differences between groups were analyzed by Student’s t-test and results were expressed as the
mean±SD.
Results
Construction of
pSUPER-sp3111-shRNA vectors
The Whitehead siRNA
(short interfering RNA) Selection Web Server
(http://jura.wi.mit.edu/bioc/siRNA) automates the design of short
oligonucleotides that can specifically ‘knock
down’ expression of target genes. And we got three
shRNA sequence of sp3111. Recombinant pSUPERsp3111- shRNA vectors were confirmed
by sequence analysis (Table 1). A 64-bp cDNA fragment had been inserted into the
expected site. Furthermore, the inserted sequence was correct. Three
recombinant plasmids of pSUPERsp3111- shRNA containing different shRNA sequence
of sp3111 were constructed successfully.
Generation of sp3111 transgenic RNAi
mice via spermatogonia-mediated gene transfer
In each case, we
electroporated the left testis of mice and surgically removed the right testis
to prevent the dilution of transgenic sperm with normal sperm. The
electroporated animals were mated with wild-type females (ratio 1:2) from 30 to
85 days and from 115 to 165 days after electroporation. Not all of the
PCR-positive offspring expressed eGFP under fluorescence microscope. The
prefounders of electroporated mice by oligo 2 generated 113 offspring (F0)
born. Thirty-eight F0 mice (33.6%) were positive as tested by PCR on tail DNA,
but only 12 pubs (10.6%) had visible eGFP expression (Table 2). Some organs
expressing eGFP under a fluorescence microscope of F1 generation
offspring were imaged (Fig. 1). A large number of samples from different transgenic
lines were determined by PCR and run on the same gel. One litter mice of F0
generation determined by PCR are shown in Fig. 2.
Real-time PCR
analysis of sp3111 mRNA expression level
The results of
real-time quantitative PCR analysis showed that the testicular expression of sp3111 gene was knocked down
at the mRNA level in transgenic male mice as compared with wild-type males. And
the efficiency of RNAi was .30% (Fig. 3).
Male sp3111 transgenic RNAi
mice have low litter sizes
The sizes of litters
from wild-type female ´ transgenic male were
significantly reduced compared with wild-type female ´ wild-type male. As shown in Fig. 4, the offspring number obtained with the
control wild-type male mice was about 11.0±1.3. But the offspring number fell dramatically to 6.0±
Discussion
One of the methods to
study a gene function is to observe the effect of the loss of function of a
gene by establishing knockout or knockdown transgenic mice. Although the
knockout technology is efficient, it is expensive, time consuming, and the
success rate is not high. RNAi has gained widespread application in gene
function research since its discovery because it is simpler, less time
consuming, and relatively inexpensive [9,10]. sp3111 transgenic RNAi mouse
model is necessary for the further exploration of the reproductive biological
function of sp3111 gene.
The ways to generate
transgenic mice are pronuclear injection [11], ES cell-based methods [12], and
lentiviralmediated transgenesis. One published paper [8] showed that
electroporation-mediated technique for transfection of undifferentiated
repopulating spermatogonial cells in vivo resulted in
integration and long-term maintenance of the transgene in the germ cell. This
method with a success rate of 94% contributes to the prolonged ability of
electroporated founders to sire transgenic progeny, in some cases, more than a
year by natural mating. It requires neither assisted reproductive techniques
nor sophisticated laboratory setup and highly trained personnel. According to
this article, we injected the linearized RNAi expressing vectors into testis,
then electroporated the testis to obtain for the first time sp3111 transgenic RNAi mouse
model by natural mating. In the sp3111 transgenic RNAi mice,
the presence of eGFP was detected by fluorescence microscopy and PCR using
genomic DNA obtained from their tail tips. While we found that the transgenic
efficiency is obviously higher in the period of 35–85 days than in the
period of 115–165 days. It indicated that the early period of
postelectroporation is the better mating time to obtain transgenic mice.
The efficiency of RNAi
is .30% by
real-time PCR analysis. The beauty of transgenic RNAi is that, unlike gene
knockouts, one does not have to breed the transgene into homozygosity to see
the effect. The model of heterozygous sp3111 transgenic RNAi mice
realized the knock down of the sp3111 expression. This remarkably shortens the time to obtain
transgenic animal. We delivered genes to the interstitium of the testes because
spermatogonial cells are located outside the blood– testis barrier at the
basement of the tubules and therefore have direct access to interstitial fluid.
Spermatogonia, which are actively proliferating and concomitantly providing
mature sperm, can be easily transfected with exogenous DNA. Notably, the
prefounders produced transgenic offspring as long as 165 days after
electroporation, indicating that the transgene was integrated into the stem
and/or transit amplifying undifferentiated spermatogonia at the time of
electroporation. Electroporated males provide a valuable resource for
continuous production of transgenic founders for as long as 165 days. Now,
fluorescent phenotype has transmitted stably to F3 generation. And we will
focus on the heredity stability of filial generation.
sp3111 transgenic RNAi mouse is necessary for in vivo study the function of sp3111. sp3111 is a membrane
protein with four transmembrane motifs, suggesting it may play roles in sperm
development and sperm–egg binding [13]. sp3111 protein might act in the process
of gamete recognition and interaction. The four transmembrane regions would
anchor the sp3111 protein in membranes of spermatozoa to carry out its
functions, such as cell communication. Also, the sp3111 protein might play an
important role in intracellular signal transduction during sperm maturation
[14,15]. The suppression of sp3111 expression may be responsible for the small litter size.
However, more work should be done to get a better understanding of its
biological function. Currently, only a few sperm-specific polypeptides have
been identified. sp3111, as one of the sperm-specific proteins, may help to
understand the fertilization and early embryo development. It can even provide
useful means in infertility and contraception. Taken together, by
electroporation-mediated technique for transfection of spermatogonial cells we
obtained sp3111 transgenic RNAi mice. It is feasible to generate
transgenic RNAi animal by this technique. And this model eliminates the need to
breed the transgenic mice into homozygosity to see the effect. The suppression
of sp3111 expression may be responsible for the small litter size. It is an ideal
model to explore the reproductive biological function of gene sp
Acknowledgements
The authors are
grateful to Prof. Yonglian Zhang (
Funding
This work was
supported by the grants from the Major State Basic Research Development Program
of China (2006GB 504005 and 2009CB941700) and the National Natural Science
Foundation of China (30872765 and 90919016).
References
1 Gadella BM. Sperm
membrane physiology and relevance for fertilization. Anim Reprod Sci 2008, 107:
229–236.
2 Kaji K and Kudo A.
The mechanism of sperm-oocyte fusion in mammals. Reproduction 2004, 127: 423–429.
3 Shi HJ, Wu AZ,
Santos M, Feng ZM and Chen CL. Identification of a novel rat
spermatid-expressing four-transmembrane gene sp3111 related to CD20 and Fc1 RIb[R]. American Society of Andrology 2004 meeting,
Baltimore, 2004.
4 Charrin S, le Naour
F, Silvie O, 5 Shi HJ, Jia XF, Zhou
M, Santos M, Wu AZ and Chen CL. Rat SP3111, a novel germ cell-expressing
four-transmembrane protein involved in early cleavage of fertilized ova. 6 Kunath T, Gish G,
Lickert H, Jones N, Pawson T and Rossant J. Transgenic RNA interference in ES
cell-derived embryos recapitulates a genetic null phenotype. Nat Biotechnol
2003, 21: 559–561.
7 Shoji M, Chuma S,
Yoshida K, Morita T and Nakatsuji N. RNA interference during spermatogenesis in
mice. Dev Biol 2005, 282: 524–534.
8 Dhup S and Majumdar
SS. Transgenesis via permanent integration of genes in repopulating
spermatogonial cells in vivo. Nature Methods 2008, 5: 601–603.
9 Song EW, Lee SK,
Wang J, Ince N, Ouyang N, Min J and Chen JS, et al. RNA interference
targeting Fas Protects mice form fulminant hepatitis. Nat Med 2003, 9: 347–351.
10 Maine EM. Studying
gene function in Caenorhabditis
elegans using RNA-mediated interference. Brief
Funct Genomic Proteomic 2008, 7: 184–194.
11 Peng SH, York JP
and Zhang PM. A transgenic approach for RNA interference-based genetic
screening in mice. Proc Natl Acad Sci USA 2006, 103: 2252–2256.
12 Carmell MA, Zhang LQ,
Conklin DS, Hannon GJ and Rosenquist TA. Germline transmission of RNAi in mice.
Nat Struct Biol 2003, 10: 91–92.
13 Zhou M, Jia XF, Shi
TY, Wang J, Yuan Y and Shi HJ. Study on the feasibility of establishing of
rsp3111 trangenic mouse through in vivo transfection of
spermatogonial stem cells. Lab Anim Comp Med 2008, 28: 138–143.
14 Zhou X, Wang J, Liu
Q, Shen J, Zheng M and Zhang YL. Identification of the gene imds15 Li XQ, Liu Q, Liu SG, Zhang JS and Zhang YL. New
member of the guanosine triphosphatase activating protein family in the human
epididymis. Acta Biochim Biophys Sin 2008, 40: 855–863.
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