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M. Stefanini - Biological characterization and in vitro culture of spermatogonial stem cells
cell heterogeneity that may be a conserved feature in
mammalian testis (Grisanti et al., submitted for publication).
In vitro Cre recombinase transduction induces
conditional gene inactivation in
spermatogonia stem cells
Gene inactivation often results in early embryonic
lethal phenotype hampering the analysis of gene
inactivation in later stages of development. To circumvents
these drawbacks, inactivation of candidate
genes have been accomplished by conditional mutagenesis.
The expression of recombinase Cre in target
cells, results in the deletion of the loxP-modified
alleles. Recently, Cre fusion proteins have been developed
that enter mammalian cells maintained in vitro
and perform recombination with high efficiency. In
order to obtain conditional mutagenesis in adult
SSCs we analyzed the feasibility of the ex vivo Cre
delivery to germ cells combined with their transplantation
in recipient mice testis. To test for Creinduced
gene recombination in SSCs, germ cells were
obtained from ROSA26 Cre reporter mice (R26R), in
which Cre recombination can be monitored by LacZ
expression. Cre-transduced SSCs retained their ability
to self-renew and differentiate in vivo. Cre-transduced
SSCs originate colonies of donor-derived
spermatogenesis, in which LacZ-expressing germ
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cells were normally arranged along seminiferous
tubules. These data demonstrated, the feasibility of
in vitro conditional mutagenesis in stem cell
germline. This experimental strategy allows genetic
manipulation of postnatal SSCs and their germ cell
progeny, and may prove useful for the study of genes
whose inactivation is either embryonically lethal or
interferes with prenatal germ cell development
(Grisanti et al., 2008).
Selected publications
Muciaccia B, Corallini S, Vicini E, Padula F, Gandini
L, Liuzzi G, Lenzi A, Stefanini M. HIV-1 viral DNA is
present in ejaculated abnormal spermatozoa of
seropositive subjects. Hum Reprod. 2007, 22:2868-78.
Puglisi R, Bevilacqua A, Carlomagno G, Lenzi A,
Gandini L, Stefanini M, Mangia F, Boitani C. Mice
overexpressing the mithochondrial phospholipid
hydroperoxide glutathione peroxidase in male germ
cells show abnormal spermatogenesis and reduced fertility.
Endocrinology 2007, 148:4302-9.
Grisanti L, Corallini S, Fera S, Muciaccia B, Witke
W, Stefanini M, Vicini E. Inactivation of numb and
numblike in spermatogonial stem cells by cell-permeant
Cre recombinase. Differentiation 2008, in press.