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1.
STAR Protoc ; 5(3): 103107, 2024 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-38963758

RESUMEN

Electroporation is a technique to introduce DNA constructs into cells using electric current. Here, we present a protocol to electroporate DNA plasmids into Ciona robusta embryos at the 1-cell stage. We describe steps for setting up and conducting electroporation. We then detail procedures for collecting, fixing, and mounting embryos and counting expression. This protocol can be used to study the expression of enhancers via reporter assays, manipulating cells using genes or modified genes such as dominant negatives, and genome editing. For complete details on the use and execution of this protocol, please refer to Song, et al.1.

2.
Dev Cell ; 58(21): 2206-2216.e5, 2023 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-37848026

RESUMEN

Transcriptional enhancers direct precise gene expression patterns during development and harbor the majority of variants associated with phenotypic diversity, evolutionary adaptations, and disease. Pinpointing which enhancer variants contribute to changes in gene expression and phenotypes is a major challenge. Here, we find that suboptimal or low-affinity binding sites are necessary for precise gene expression during heart development. Single-nucleotide variants (SNVs) can optimize the affinity of ETS binding sites, causing gain-of-function (GOF) gene expression, cell migration defects, and phenotypes as severe as extra beating hearts in the marine chordate Ciona robusta. In human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a SNV within a human GATA4 enhancer increases ETS binding affinity and causes GOF enhancer activity. The prevalence of suboptimal-affinity sites within enhancers creates a vulnerability whereby affinity-optimizing SNVs can lead to GOF gene expression, changes in cellular identity, and organismal-level phenotypes that could contribute to the evolution of novel traits or diseases.


Asunto(s)
Elementos de Facilitación Genéticos , Células Madre Pluripotentes Inducidas , Humanos , Elementos de Facilitación Genéticos/genética , Miocitos Cardíacos/metabolismo , Sitios de Unión , Nucleótidos
3.
Cell Rep ; 42(2): 112052, 2023 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-36729834

RESUMEN

The notochord is a defining feature of all chordates. The transcription factors Zic and ETS regulate enhancer activity within the notochord. We conduct high-throughput screens of genomic elements within developing Ciona embryos to understand how Zic and ETS sites encode notochord activity. Our screen discovers an enhancer located near Lama, a gene critical for notochord development. Reversing the orientation of an ETS site within this enhancer abolishes expression, indicating that enhancer grammar is critical for notochord activity. Similarly organized clusters of Zic and ETS sites occur within mouse and human Lama1 introns. Within a Brachyury (Bra) enhancer, FoxA and Bra, in combination with Zic and ETS binding sites, are necessary and sufficient for notochord expression. This binding site logic also occurs within other Ciona and vertebrate Bra enhancers. Collectively, this study uncovers the importance of grammar within notochord enhancers and discovers signatures of enhancer logic and grammar conserved across chordates.


Asunto(s)
Ciona intestinalis , Notocorda , Animales , Humanos , Ratones , Ciona intestinalis/genética , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismo , Regulación del Desarrollo de la Expresión Génica , Elementos de Facilitación Genéticos/genética
4.
Hepatol Commun ; 5(10): 1649-1659, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34558837

RESUMEN

Integrated hepatitis B virus (HBV) DNA, found in more than 85% of HBV-associated hepatocellular carcinomas (HBV-HCCs), can play a significant role in HBV-related liver disease progression. HBV-host junction sequences (HBV-JSs), created through integration events, have been used to determine HBV-HCC clonality. Here, we investigate the feasibility of analyzing HBV integration in a noninvasive urine liquid biopsy. Using an HBV-targeted next-generation sequencing (NGS) assay, we first identified HBV-JSs in eight HBV-HCC tissues and designed short-amplicon junction-specific polymerase chain reaction assays to detect HBV-JSs in matched urine. We detected and validated tissue-derived junctions in five of eight matched urine samples. Next, we screened 32 urine samples collected from 25 patients infected with HBV (5 with hepatitis, 10 with cirrhosis, 4 with HCC, and 6 post-HCC). Encouragingly, all 32 urine samples contained HBV-JSs detectable by HBV-targeted NGS. Of the 712 total HBV-JSs detected in urine, 351 were in gene-coding regions, 11 of which, including TERT (telomerase reverse transcriptase), had previously been reported as recurrent integration sites in HCC tissue and were found only in the urine patients with cirrhosis or HCC. The integration breakpoints of HBV DNA detected in urine were found predominantly (~70%) at a previously identified integration hotspot, HBV DR1-2 (down-regulator of transcription 1-2). Conclusion: HBV viral-host junction DNA can be detected in urine of patients infected with HBV. This study demonstrates the potential for a noninvasive urine liquid biopsy of integrated HBV DNA to monitor patients infected with HBV for HBV-associated liver diseases and the efficacy of antiviral therapy.


Asunto(s)
Carcinoma Hepatocelular/orina , ADN Viral/orina , Virus de la Hepatitis B/genética , Neoplasias Hepáticas/orina , Integración Viral/genética , Adulto , Anciano , Sitios de Ligazón Microbiológica/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virología , ADN Viral/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa
5.
J Mol Diagn ; 14(2): 112-9, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22251609

RESUMEN

We demonstrated previously that urine contains low-molecular-weight (LMW) (<300 bp), circulation-derived DNA that can be used to detect cancer-specific mutations if a tumor is present. The goal of this study was to develop an assay to detect the colorectal cancer (CRC)-associated, circulation-derived, epigenetic DNA marker hypermethylated vimentin gene (mVIM) in the urine of patients with CRC. An artificial 18-nucleotide DNA sequence was tagged at the 5' end of the primers of the first PCR cycle to increase the amplicon size, which was then integrated into the primers of the second PCR cycle. A quantitative MethyLight PCR-based assay targeting a 39-nucleotide template was developed and used to quantify mVIM in CRC tissues and matched urine samples. mVIM was detected in 75% of LMW urine DNA samples from patients with CRC (n = 20) and in 10% of urine samples of control subjects with no known neoplasia (n = 20); 12 of 17 LMW urine DNA samples (71%) but only 2 of 17 high-molecular-weight urine DNA samples (12%) from patients with mVIM-positive tissues contained detectable mVIM, suggesting that the mVIM detected in LMW urine DNA is derived from the circulation. The detection of mVIM in urine was significantly associated with CRC compared with controls (P < 0.0001, by Fisher's exact test). A potential urine test for CRC screening using epigenetic markers is discussed.


Asunto(s)
Biomarcadores de Tumor/orina , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/orina , Metilación de ADN , ADN de Neoplasias/orina , Vimentina/genética , Vimentina/orina , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , ADN de Neoplasias/genética , Epigenómica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Neoplásicas Circulantes/patología , Reacción en Cadena en Tiempo Real de la Polimerasa
6.
J Neurovirol ; 16(5): 384-98, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20874012

RESUMEN

Using polymerase chain reaction (PCR) and alkaline gel electrophoresis, the authors show that, compared with DNA derived from virions used to establish infection, herpes simplex virus DNA derived from quiescently infected rat pheochromocytoma (PC12) cells in culture accumulates alkaline-labile lesions. That is, compared with equivalent amounts of virion DNA, viral DNA from nerve growth factor-differentiated long-term infected cells in culture is consistently 3 to 10 times more refractory to amplification by PCR. Despite using equal mole amounts of DNA isolated from quiescently infected cells (determined by quantitative Southern blots), DNA from quiescently infected cells could not be detected by PCR under conditions in which the virion-derived DNA was easily detected. Refractoriness to PCR was confirmed by analysis with a ligation-mediated PCR technique. The refractoriness was not the result of genomic circularization. The refractoriness was, however, related to the time that the quiescently infected cells had been maintained in culture. The refractoriness to PCR was taken as an indication that the viral DNA was damaged. This hypothesis was confirmed by showing that viral DNA from quiescently infected PC12 cells accumulated alkaline-labile DNA lesions, as determined by alkaline gel electrophoresis. The phenomenon was not limited to tissue culture, because viral DNA derived from the ganglia of latently infected mice is also 3 to 10 times more refractory to amplification than are equivalent amounts of virion-derived genomes. Taken together, these results represent the first evidence that herpes simplex virus DNA is physically damaged as a function of long-term infection. Implications for viral reactivation and pathogenesis are discussed.


Asunto(s)
Daño del ADN , ADN Viral/genética , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Neuronas/virología , Latencia del Virus/genética , Animales , ADN Viral/aislamiento & purificación , Femenino , Herpesvirus Humano 1/genética , Ratones , Ratones Endogámicos BALB C , Células PC12 , Reacción en Cadena de la Polimerasa , Ratas , Factores de Tiempo
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