Targeting genes for self-excision in the germ line

Bunting, Michaeline; Bernstein, Kenneth E.; Greer, Joy M.; Capecchi, Mario R.; Thomas, Kirk R.

doi:10.1101/gad.13.12.1524

Cited by 229 publications

(198 citation statements)

References 27 publications

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“…A self-excision neomycinresistance (neo R ) cassette, in which a phosphoglycerate kinase (PGK) promoter-driven neomycin-resistance marker and an angiotensin-converting enzyme (ACE) promoter-driven yeast FLP recombinase gene were placed in between a loxP and frt recombination site on one side and a second frt site on the other side, was constructed by multiple steps of PCR-based cloning. The PGK-neo R region was amplified from pPGKNeo͞TK2 (8), the ACE-FLP region from pACN (12) and pFLPe (13), and the frt sites from pPRT2 (14). The design of this cassette followed that reported in ref.…”

Section: Methodsmentioning

confidence: 99%

Aberrant lamination in the cerebral cortex of mouse embryos lacking DNA topoisomerase IIβ

Lyu

Wang²

2003

Proc. Natl. Acad. Sci. U.S.A.

106

115

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We have examined corticogenesis in mouse embryos lacking DNA topoisomerase II␤ (II␤) in the brain or in all tissues. The absence of II␤, a type II DNA topoisomerase normally expressed in postmitotic cells in the developing cortex, severely affects cerebral stratification: no subplate is discernible, and neurons born at later stages of corticogenesis fail to migrate to the superficial layers. This abnormal pattern of neuron positioning in the cerebral cortex is reminiscent of that observed in mouse mutants defective in the reelin-signaling pathway. Significantly, the level of reelin in the neocortex is much reduced when II␤ is absent. These results implicate a role of II␤ in brain development. The enzyme may be required in implementing particular genetic programs in postmitotic cells, such as reelin expression in Cajal-Retzius cells, perhaps through its action on nucleoprotein structure of particular chromosomal regions.M ammalian DNA topoisomerase II␤ (II␤) is a type II DNA topoisomerase that catalyzes the transport of one DNA double-helix through another (1-3). Unlike the II␣ isozyme, which is specifically expressed in proliferating cells and most likely performs the essential function of resolving intertwined chromosome pairs during mitosis (4-6), II␤ is apparently dispensable in cell growth (reviewed in ref. 7). Targeted disruption of the murine TOP2␤ gene showed; however, that II␤ has a critical role in neural and neuromuscular development (8). Whereas top2␤ ϩ/⌬ mice are phenotypically TOP2␤ ϩ and indistinguishable from their TOP2␤ ϩ/ϩ littermates, top2␤ ⌬/⌬ nullizygotes die of a breathing impairment at birth, most likely owing to neural and neuromuscular defects (8).In mammals, II␣ and II␤ are largely expressed in proliferating cells and nonproliferating cells, respectively (7). In a detailed examination of the expression of the two forms during postnatal development of rat cerebellum, a sharp transition from II␣ to II␤ expression was observed in cell populations that had undergone the final division and committed to differentiate into Purkinje or granule cells (9). The amount of DNA-bound II␤ becomes undetectable, however, when cells reach terminal differentiation (10).To further test the role of II␤ in neural development, we have examined cerebral corticogenesis in embryos of mouse lines lacking II␤ in all tissues or specifically in certain brain structures including the telencephalon. We report here that II␤ has a remarkable role in neurogenesis. The induction of II␤ expression, after the final division of cells committed to a neuronal fate, appears necessary for the developmentally regulated expression of certain specific genes in these cells. The role of II␤ is unlikely to be limited to neuronal development and may also be important in the genetic programming of other postmitotic cells. Materials and MethodsConstruction of the Targeting Vector. The left and right arms of TOP2␤-derived sequences, for targeted insertion of DNA sequences between them into the mouse genome, were obtained by PCR. The right ...

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Section: Methodsmentioning

confidence: 99%

Aberrant lamination in the cerebral cortex of mouse embryos lacking DNA topoisomerase IIβ

Lyu

Wang²

2003

Proc. Natl. Acad. Sci. U.S.A.

106

115

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“…Generation and breeding of mouse lines, tamoxifen treatment and detection of floxed and recombined alleles A K14-Cre transgene was made by inserting a modified Cre cDNA fragment PCR-amplified from the pACN vector (Bunting et al, 1999) into the BamHI site of a K14 expression vector (Andl et al, 2002). The transgene was microinjected into fertilized eggs from a B6SJLF1/J×B6SJLF1/J cross (Jackson Laboratories).…”

Section: Methodsmentioning

confidence: 99%

EpithelialBmpr1aregulates differentiation and proliferation in postnatal hair follicles and is essential for tooth development

et al. 2004

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Bone morphogenetic protein (BMP) signaling is thought to perform multiple functions in the regulation of skin appendage morphogenesis and the postnatal growth of hair follicles. However, definitive genetic evidence for these roles has been lacking. Here, we show that Cre-mediated mutation of the gene encoding BMP receptor 1A in the surface epithelium and its derivatives causes arrest of tooth morphogenesis and lack of external hair. The hair shaft and hair follicle inner root sheath (IRS) fail to differentiate, and expression of the known transcriptional regulators of follicular differentiation Msx1,Msx2, Foxn1 and Gata3 is markedly downregulated or absent in mutant follicles. Lef1 expression is maintained, but nuclearβ-catenin is absent from the epithelium of severely affected mutant follicles, indicating that activation of the WNT pathway lies downstream of BMPR1A signaling in postnatal follicles. Mutant hair follicles fail to undergo programmed regression, and instead continue to proliferate, producing follicular cysts and matricomas. These results provide definitive genetic evidence that epithelial Bmpr1a is required for completion of tooth morphogenesis, and regulates terminal differentiation and proliferation in postnatal hair follicles.

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“…To generate CA IV KO mice, we constructed a replacement targeting vector by ligating a 3.6-kb KpnI-XhoI fragment, containing intron 1a of the CA 4 gene, on one side of the Neo gene of the targeting vector pPNT-Cass LoxA (16,17) and a 4.5-kb XhoI-NotI genomic fragment, extending from exon 4 to intron 7, on the other side of the Neo gene. Deleted exons 1b, 2, and 3, and 9 aa of exon 4 encoded all three active-site His residues in the 146 aa immediately after the signal sequence.…”

Section: Methodsmentioning

confidence: 99%

Carbonic anhydrase IV and XIV knockout mice: Roles of the respective carbonic anhydrases in buffering the extracellular space in brain

Shah

Ulmasov

Waheed

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

113

118

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Previous studies have implicated extracellular carbonic anhydrases (CAs) in buffering the alkaline pH shifts that accompany neuronal activity in the rat and mouse hippocampus. CAs IV and XIV both have been proposed to mediate this extracellular buffering. To examine the relative importance of these two isozymes in this and other physiological functions attributed to extracellular CAs, we produced CA IV and CA XIV knockout (KO) mice by targeted mutagenesis and the doubly deficient CA IV͞XIV KO mice by intercrossing the individual null mice. Although CA IV and CA XIV null mice both are viable, the CA IV nulls are produced in smaller numbers than predicted, indicating either fetal or postnatal losses, which preferentially affect females. CA IV͞XIV double KO mice are also produced in fewer numbers than predicted and are smaller than WT mice, and many females die prematurely before and after weaning. Electrophysiological studies on hippocampal slices on these KO mice showed that either CA can mediate buffering after synaptic transmission in hippocampal slices in the absence of the other, but that eliminating both is nearly as effective as the CA inhibitor, benzolamide, in blocking the buffering seen in the WT mice. Thus, both CA IV and CA XIV contribute to extracellular buffering in the central nervous system, although CA IV appears to be more important in the hippocampus. These individual and double KO mice should be valuable tools in clarifying the relative contributions of each CA to other physiological functions where extracellular CAs have been implicated. interstitial pH ͉ mouse hippocampus ͉ targeted mutagenesis

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Targeting genes for self-excision in the germ line

Cited by 229 publications

References 27 publications

Aberrant lamination in the cerebral cortex of mouse embryos lacking DNA topoisomerase IIβ

Aberrant lamination in the cerebral cortex of mouse embryos lacking DNA topoisomerase IIβ

EpithelialBmpr1aregulates differentiation and proliferation in postnatal hair follicles and is essential for tooth development

Carbonic anhydrase IV and XIV knockout mice: Roles of the respective carbonic anhydrases in buffering the extracellular space in brain

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