DNA methylation pattern of a tandemly repeatedLacZ transgene indicates that most copies are silent

Lau, Stephen; Jardine, Karen; McBurney, Michael W.

doi:10.1002/(sici)1097-0177(199906)215:2<126::aid-dvdy5>3.0.co;2-8

Cited by 22 publications

(2 citation statements)

References 40 publications

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“…The inclusion of insulated monomers will show how MAR-elements [ 103 , 132 ] behave in a silenced concatemer. Since the order of copies in the repeat could have an effect on expression [ 133 ], it would be interesting to examine if the flanking copies avoid silencing. During concatenation, transgene copies are joined randomly, and the distribution of monomer variants in multicopy integrants will depend on the proportion of monomers in the delivered DNA ( Figure 5 ).…”

Section: Repeat-induced Gene Silencingmentioning

confidence: 99%

Concatenation of Transgenic DNA: Random or Orchestrated?

Smirnov

Battulin

2021

Genes

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Generation of transgenic organisms by pronuclear microinjection has become a routine procedure. However, while the process of DNA integration in the genome is well understood, we still do not know much about the recombination between transgene molecules that happens in the first moments after DNA injection. Most of the time, injected molecules are joined together in head-to-tail tandem repeats—the so-called concatemers. In this review, we focused on the possible concatenation mechanisms and how they could be studied with genetic reporters tracking individual copies in concatemers. We also discuss various features of concatemers, including palindromic junctions and repeat-induced gene silencing (RIGS). Finally, we speculate how cooperation of DNA repair pathways creates a multicopy concatenated insert.

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Section: Repeat-induced Gene Silencingmentioning

confidence: 99%

Concatenation of Transgenic DNA: Random or Orchestrated?

Smirnov

Battulin

2021

Genes

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“…No significant variation of lacZ expression was observed in the five progeny animals from each line ( Table II), suggesting that once established as a line, transgene expression is stably maintained. In contrast, ␤-gal ϩ neurons were not found in the other (24). The Core Sequence within OE1 Is Essential for Prepro-orexin Gene Expression-To identify the core region of OE1 that specifies prepro-orexin gene expression in the LHA, we prepared several deletion constructs of OE1 (Ϫ287 to Ϫ77 bp) and examined them in a series of transgenic founder mouse assays (Fig.…”

Section: And Below)mentioning

confidence: 99%

The Human Prepro-orexin Gene Regulatory Region That Activates Gene Expression in the Lateral Region and Represses It in the Medial Regions of the Hypothalamus

Moriguchi¹,

Sakurai²,

Takahashi³

et al. 2002

Journal of Biological Chemistry

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Prepro-orexin is a precursor of the neuropeptides orexin-A and -B, which are localized in the neuronal population of the lateral hypothalamic area (LHA). We wished to elucidate the mechanisms by which the prepro-orexin gene is specifically activated in orexin neurons in the LHA. The 3.2-kb 5-flanking region of the human prepro-orexin gene is sufficient for the specific expression of an Escherichia coli lacZ reporter gene in orexin neurons. Therefore, we examined a series of reporter constructs harboring this 3.2-kb regulatory region or its deletion in a reporter transgenic mouse assay. There are two phylogenetically conserved regions located 287 bp (orexin regulatory element (OE) 1) and 2.5 kb (OE2) upstream of the transcription initiation site of the human prepro-orexin gene. In transgenic mice, both OE1 and OE2 are necessary for expressing the human prepro-orexin gene in the LHA and for repressing its expression in the medial regions of the hypothalamus. Through serial deletion analysis of OE1, we found that the 57-bp core region of OE1 is critical for its spatial gene regulatory function in vivo. Mutation analysis further demonstrated that without contribution from the OE1 core region, the lacZ reporter is expressed ectopically in the medial regions of the hypothalamus. Thus, OE1 contains crucial cis-acting elements regulating prepro-orexin gene expression specifically in the LHA.Regulation of food intake and energy homeostasis takes place mainly in the medial regions of the hypothalamus, where many of the necessary neuropeptides exist (1, 2). In the mediobasal hypothalamus, the arcuate nucleus (ARC) 1 houses neurons expressing neuropeptide Y, ␣-melanocyte-stimulating hormone, and agouti-related peptide, which are critical in modulating body weight and metabolism (3, 4). Several lateral hypothalamic neuropeptides controlling the regulation of energy balance and food intake have also been identified (5). For instance, orexin-A (hypocretin-1) and orexin-B (hypocretin-2) and their common precursor peptide prepro-orexin are specifically localized in neurons located in the lateral hypothalamic area (LHA), a region classically regarded as the "feeding center" (6 -8). These neuropeptides are ligands for two closely related orphan G-protein-coupled receptors, OX1r and OX2r (8, 9). OX1r is selective for orexin-A, whereas nonspecific OX2r binds both isopeptides. In rats, intracerebroventricular administration of orexins stimulates food consumption, whereas the expression level of mRNA is up-regulated by fasting (8) and decreased in genetically obese ob and db mice (10). The expression level of the prepro-orexin gene is regulated by leptin and orexin-containing neurons express the leptin receptor (11).It is believed that orexins also play roles in regulating states of arousal, sleep, and wakefulness. For example, it was reported that targeted disruption of the mouse prepro-orexin gene results in a sleeping disorder strikingly similar to human narcolepsy (12). Furthermore, mutations of the OX2 gene were found in a canine...

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Reexpression of a cluster of silenced transgenes is associated with their rearrangement

McBurney

Lau

Jardine

et al. 2001

Genes Chromosomes & Cancer

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Irreversible inactivation or silencing of tumor suppressor genes occurs frequently in the development of cancer. A similar process of silencing can occur after the integration of transfected or microinjected genes into the genomes of recipient cells. The inactivation of transfected genes seems particularly efficient in cells with stem cell characteristics. We have been studying the inactivation of genes transfected into cultured P19 embryonal carcinoma cells and found that the CpG-rich sequence comprising the coding region of the lacZ reporter gene becomes extensively methylated after integration into the genome. 5-Aza-2'-deoxycytidine (5AdC), an inhibitor of DNA methylation, induced the reexpression of silent transgenes in one clone of P19 cells studied in detail. However, the reexpressed genes remained heavily methylated over the lacZ coding sequence. We used pulsed-field gel electrophoresis to analyze the structure of the transgenic locus in the parental and in 5AdC-treated cells and found that, in each of the cells reexpressing the transgene, the cluster of transgenes had been rearranged. Each clone had undergone a different rearrangement that appeared to involve recombination within the tandemly repeated copies of the transgene. Our data seem consistent with the idea that 5AdC induces efficient DNA recombination between tandemly repeated genes and that the reexpression of silenced genes induced by 5AdC might be triggered by the chromatin reorganization at the site of DNA recombination.

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DNA methylation pattern of a tandemly repeatedLacZ transgene indicates that most copies are silent

Cited by 22 publications

References 40 publications

Concatenation of Transgenic DNA: Random or Orchestrated?

Concatenation of Transgenic DNA: Random or Orchestrated?

The Human Prepro-orexin Gene Regulatory Region That Activates Gene Expression in the Lateral Region and Represses It in the Medial Regions of the Hypothalamus

Reexpression of a cluster of silenced transgenes is associated with their rearrangement

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