GFP reporter mice for the retinoblastoma-related cell cycle regulator p107

Burkhart, Deborah L.; Viatour, Patrick; Ho, Victoria M.; Sage, Julien

doi:10.4161/cc.7.16.6441

Cited by 9 publications

(16 citation statements)

References 52 publications

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“…28,29 It has been shown that, in the absence of pRb, negative cell growth genes, including other Rb family members, may be upregulated to compensate for pRb function. [30][31][32] Correlating with this, microarray analysis showed upregulation of Rbl1 (p107), Cdkn1a (p21 Cip1 ) and Cdkn2d (p19 Ink4d ) in the pRb -/-inner ear.…”

Section: Resultsmentioning

confidence: 99%

Overlapping and distinct pRb pathways in the mammalian auditory and vestibular organs

Huang

Sage²,

Tang³

et al. 2011

Cell Cycle

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Section: Resultsmentioning

confidence: 99%

Overlapping and distinct pRb pathways in the mammalian auditory and vestibular organs

Huang

Sage²,

Tang³

et al. 2011

Cell Cycle

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“…Mice were euthanized 3 days after the last injection. In unpublished observations related to a previous study (8), we found that injection of tamoxifen alone in Rosa26…”

Section: Rosa26mentioning

confidence: 92%

“…Finally, the Rb mRNA may be subject to posttranscriptional regulation that does not affect the eGFP mRNA, or the eGFP mRNA could be more stable than the Rb mRNA. We believe that a differential regulation of the Rb mRNA is more likely, because we recently described the use of a similar BAC transgenic reporter for the Rb family member p107 and did not observe any unusual stability or instability of the RNA from the same eGFP cassette in the liver or other organs (except the testes) of these transgenic mice (8). Posttranscriptional regulation of Rb may explain why eGFP is expressed at higher levels in the adult kidney than is Rb, as the eGFP transcript is missing any 3Ј UTR sequences that could be regulated by endogenous microRNAs.…”

Section: Discussionmentioning

confidence: 99%

“…Recombineering was used to generate the Rb-eGFP BAC, as described previously (8). Briefly, BAC clone RP24-370G12, ordered from BACPAC (http://bacpac .chori.org/), was transformed into the recombineering strain EL250.…”

Section: Rosa26mentioning

confidence: 99%

“…For cell cycle genes, SYBR GreenER master mix (Invitrogen) was used. GFP and p107 primer/probes were described previously (8). All relative expression analyses were calculated relative to TBP (TATA binding protein) expression.…”

Section: Creert2mentioning

confidence: 99%

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Regulation of RB Transcription In Vivo by RB Family Members

Burkhart¹,

Ngai²,

Roake³

et al. 2010

Molecular and Cellular Biology

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In cancer cells, the retinoblastoma tumor suppressor RB is directly inactivated by mutation in the RB gene or functionally inhibited by abnormal activation of cyclin-dependent kinase activity. While variations in RB levels may also provide an important means of controlling RB function in both normal and cancer cells, little is known about the mechanisms regulating RB transcription. Here we show that members of the RB and E2F families bind directly to the RB promoter. To investigate how the RB/E2F pathway may regulate Rb transcription, we generated reporter mice carrying an eGFP transgene inserted into a bacterial artificial chromosome containing most of the Rb gene. Expression of eGFP largely parallels that of Rb in transgenic embryos and adult mice. Using these reporter mice and mutant alleles for Rb, p107, and p130, we found that RB family members modulate Rb transcription in specific cell populations in vivo and in culture. Interestingly, while Rb is a target of the RB/E2F pathway in mouse and human cells, Rb expression does not strictly correlate with the cell cycle status of these cells. These experiments identify novel regulatory feedback mechanisms within the RB pathway in mammalian cells.The RB tumor suppressor gene was first identified through its direct mutation or deletion in human retinoblastoma. The RB protein is thought to function largely as a transcriptional cofactor that can repress or potentiate the functions of numerous transcription factors, affecting the expression of a broad number of target genes. Since its initial discovery, RB function has been demonstrated to be inactivated in virtually all human cancers through a variety of mechanisms. In particular, in addition to direct mutation events in the RB gene, the RB protein is often functionally inactivated by phosphorylation in tumor cells with constitutive activation of cyclin/Cdk complexes (44,55). RB inactivation through reduced transcription may also participate in the development of cancer (4, 41); surprisingly, however, little is known about the mechanisms regulating RB transcription in normal and tumor cells.Interestingly, the RB promoter contains a conserved binding site for the E2F transcription factors, some of which are direct partners of RB and key downstream mediators of RB (9). Increasing evidence suggests that this site may contribute to the regulation of RB transcription. For instance, overexpression of E2F1 can activate RB transcription, which may contribute to the variations in RB mRNA levels during cell cycle progression that are observed in some contexts (30, 43) but not in others (5,13,20). In addition, methylation of the E2F site in the RB promoter is sufficient to recruit repressor complexes to inhibit RB transcription (15). Accordingly, reporter assays show that mutation of the E2F site in the RB promoter leads to an absence of RB repression (33). Furthermore, mutation of the E2F (and Sp1) sites in the mouse Rb promoter in transgenic mice has revealed the importance of these sites for Rb expression in vivo (1).Togeth...

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