Transgenic mice exhibiting inducible and spontaneous Cre activities driven by a bovine keratin 5 promoter that can be used for the conditional analysis of basal epithelial cells in multiple organs

Liang et al. (2009). J Biomed Sci DOI: 10.1186/1423-0127-16-2 Citations: 31

K5-Cre and K14-Cre mice recombined through the female germ line. A 5.2-kb bovine K5 promoter driving CreERT gave one 4-OHT-inducible and one spontaneous line. Both marked basal epithelia in several organs in R26R mice, and only the spontaneous line marked mammary gland.

Parts used

BK5 PromoterSV40 Late PolyA Signal

Key findings

  • A transgene built from a 5.2-kb bovine keratin 5 (K5) promoter, a 0.5-kb intron, a Cre-ERT fusion (Cre recombinase fused to a mutated estrogen receptor ligand-binding domain), an SV40 polyadenylation signal and two copies of the ~1.2-kb chicken β-globin 5′ HS4 insulator (a barrier element against chromosomal position effects) forms a 10.8-kb BK5-CreERT construct. Pronuclear microinjection of BK5-CreERT yields four fertile transgenic founders on a C57BL/6J background, and three of the four lines show 4-hydroxytamoxifen (4-OHT)-induced Cre recombinase activity in crosses to ROSA26 Cre reporter (R26R) mice.
  • In Tg(BK5-CreERT)I;R26R mice, three intraperitoneal doses of 4-OHT (4–5 mg/kg body weight, every other day) induce Cre-mediated LacZ reporter activation, detected by X-gal staining, in the K5-expressing basal epithelia of the epidermis and its derivatives, the esophageal and foregastric mucosa, the cervicovaginal epithelia of the female reproductive tract and the medullary thymic epithelium. Vehicle-treated Tg(BK5-CreERT)I;R26R controls are used to show the 4-OHT dependence, and non-specific β-galactosidase activity appears in the hindstomach.
  • The Tg(BK5-CreERT)S line, which carries the same BK5-CreERT transgene as Tg(BK5-CreERT)I, shows Cre activity without 4-OHT, and the mechanism triggering this spontaneous activity is unclear. In Tg(BK5-CreERT)S;R26R embryos, X-gal-positive cells first appear at around embryonic day 14.5 in the epidermis, whisker follicles and hair follicles, and at birth the esophagus and forestomach, but not the hindstomach, are strongly stained.
  • In Tg(BK5-CreERT)S;R26R mice, spontaneous Cre reporter activity in the thymic medulla is undetectable at postnatal day 1 and appears only as scattered X-gal-positive foci by postnatal day 7, although endogenous K5 is expressed in the thymic epithelium during development. The expression timing of BK5-CreERT in the thymus therefore does not match endogenous K5 expression during organogenesis.
  • Tg(BK5-CreERT)S females do not act as female germ-line Cre deleters, unlike the previously described K5-Cre and K14-Cre mice that express Cre in oocytes. Offspring of R26R/R26R males crossed to Tg(BK5-CreERT)S/+ females keep K5-lineage-restricted X-gal staining in the epidermis, hair follicles, sebaceous cells and basal cells of the tracheal pseudostratified epithelium, so breeding schemes for tissue-specific recombination with Tg(BK5-CreERT)S need not consider whether the male or female carries the transgene.
  • Tg(BK5-CreERT)S;R26R females show spontaneous Cre recombination in the mammary gland, with X-gal-positive basal and luminal epithelial cells at postnatal day 7, in virgins and during lactation, whereas Tg(BK5-CreERT)I mice do not show mammary Cre-mediated recombination. The BK5-CreERT transgene in the Tg(BK5-CreERT)S line therefore reports recombination in K5-expressing basal epithelium and in luminal epithelium of the mammary gland.
  • In the prostate of Tg(BK5-CreERT)S;R26R mice, spontaneous Cre reporter activity is confined to the basal compartment of the anterior prostate lobes but occurs in both basal and luminal epithelium of the lateral prostate lobes. Cre activity from the BK5-CreERT transgene is therefore present in only a subpopulation of the epithelial cells of these glands.
  • In the urogenital system of Tg(BK5-CreERT)S;R26R females at postnatal day 7, spontaneous Cre reporter activity is strong in the transitional epithelium lining the bladder and in the stratified epithelium lining the sinus vagina, with very few X-gal-positive epithelial cells in the Müllerian vagina. The difference is possibly due to more advanced epithelial stratification in the sinus vagina than in the Müllerian vagina, which starts as simple columnar epithelium.

Abstract

BACKGROUND: Cre/loxP-mediated genetic modification is the most widely used conditional genetic approach used in the mouse. Engineered Cre and the mutated ligand-binding domain of estrogen receptor fusion recombinase (CreERT) allow temporal control of Cre activity. RESULTS: In this study, we have generated two distinct transgenic mouse lines expressing CreERT, which show 4-hydroxytamoxifen (4-OHT)-inducible and spontaneous (4-OHT-independent) Cre activities, referred to Tg(BK5-CreERT)I and Tg(BK5-CreERT)S, respectively. The transgenic construct is driven by the bovine Keratin 5 promoter, which is active in the basal epithelial lineage of stratified and pseudo-stratified epithelium across multiple organs. Despite the difference in 4-OHT dependency, the Tg(BK5-CreERT)I and Tg(BK5-CreERT)S mouse lines shared similar Cre-mediated recombination among various organs, except for unique mammary epithelial Cre activity in Tg(BK5-CreERT)S females. CONCLUSION: These two new transgenic mouse lines for the analysis of basal epithelial function and for the genetic modification have been created allowing the identification of these cell lineages and analysis of their differentiation during embryogenesis, during perinatal development and in adult mice.