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Bmi1 plays an essential part in the self-renewal of hematopoietic and neural stem cells. To investigate its role in other adult stem cell populations, we generated a mouse expressing a tamoxifen-inducible Cre from the Bmi1 locus. We found that Bmi1 is expressed in discrete cells located near the bottom of crypts in the small intestine, predominantly four cells above the base of the crypt (+4 position). Over time, these cells proliferate, expand, self-renew and give rise to all the differentiated cell lineages of the small intestine epithelium. The induction of a stable form of b-catenin in these cells was sufficient to rapidly generate adenomas. Moreover, ablation of Bmi1 + cells using a Rosa26 conditional allele, expressing diphtheria toxin, led to crypt loss. These experiments identify Bmi1 as an intestinal stem cell marker in vivo. Unexpectedly, the distribution of Bmi1-expressing stem cells along the length of the small intestine suggested that mammals use more than one molecularly distinguishable adult stem cell subpopulation to maintain organ homeostasis.Adult somatic stem cells are defined by two major properties: the ability to generate more stem cells (self-renewal) and the ability to generate differentiated cell lineages 1 . To establish the presence of these two properties, the gold standard is to assess both of them in vivo and in vitro 2 . Several studies have shown in vitro that many tissues carry cells capable of selfrenewal and of giving rise to differentiated cell types. However, few experiments have clearly established in vivo self-renewal and multipotency of these cells over time 3,4 .To address this, we established a genetic fate-mapping system for stem cell populations in vivo. We chose as our target locus Bmi1, a gene already known to be involved in the selfrenewal of neuronal, hematopoietic and leukemic cells [5][6][7] . Bmi1 was first identified in a mouse proviral insertion screen for lymphomagenesis 8 . It is part of the Polycomb group gene family, and specifically a member of polycomb-repressing complex 1 (PRC1). PRC1 has an essential role in maintaining chromatin silencing 9, 10. Bmi1 −/− mice die before or near weaning from a defect in self-renewal of hematopoietic stem cells 11,12 . Our hypothesis was that PRC1 might be part of a general mechanism for maintaining self-renewal in various adult stem cell populations.We inserted a construct encoding a internal ribosome entry site (IRES)-Cre-estrogen receptor binding domain fusion (IRES-Cre-ER) into the 3′ untranslated region of Bmi1 ( Supplementary Fig. 1 To evaluate the repopulation kinetics of the crypt cells derived from Bmi1 + parents, we analyzed the small intestines of Bmi1 Cre-ER/+ ;Rosa26 LacZ/+ mice from day 2 to day 30 after tamoxifen induction, analyzing and following crypts on serial adjacent sections. Crypts with LacZ + cells presented a large variation in the number of stained cells. From day 2 to day 30, there was a continual increase in the number of cells labeled per crypt . Over time, the number of crypts th...

Homologous recombination between DNA sequences residing in the chromosome and newly introduced, cloned DNA sequences (gene targeting) allows the transfer of any modification of the cloned gene into the genome of a living cell. This article discusses the current status of gene targeting with particular emphasis on germ line modification of the mouse genome, and describes the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred.