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Cell Lineage Analysis: X-Inactivation Mosaics

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In female mammals, one of the two X chromosomes in each and every embryonic cell is randomly inactivated during embryogenesis, leaving only one active X chromosome per cell and thereby maintaining dosage parity with males (1 ). This natural phenomenon, known as X inactivation or lyonization, provides an ideal nonsurgical method of producing mosaicism among otherwise identical cells of the embryo. We have created a line of mice (H253) by pronuclear injection of a DNA fragment containing the lacZ reporter gene under the control of an ubiquitously active promoter (HMG CoA reductase; see ref. 2 ). Breeding and chromosome hybridization experiments (FISH) with line H253 confirmed that the lacZ gene is inserted into the X-chromosome (3 ,4 ). Male members of this line express the X-linked lacZ gene in all cells of the embryo, including the developing nervous system (5 ). Ubiquitous expression of the lacZ gene produces a nuclear-localized β-gal protein, which, after histochemical reaction with X-gal substrate, is visualized as a blue reaction product visible in the cell nuclei in whole-mount embryos, organs, or tissue sections. In hemizygous females, only one of the two Xs carry the lacZ transgene and this lacZ -bearing chromosome will be randomly turned off in approx 50% of cells (3 ). The marking process is indelible and heritable (Fig. 1A ). The lacZ is integrated in the genome and every time a cell divides, the lacZ gene and either its active or inactive status is passed onto its progeny, forming a marked clone. The initial process of random inactivation occurs early in embryogenesis and is virtually completed by 9.5 d of gestation (E9.5) before organogenesis commences for most tissues.
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