RESUMO
Genetic manipulation via transgene overexpression, RNAi, or Cas9-based methods is central to biomedical research. Unfortunately, use of these tools is often limited by vector options. We have created a modular platform (pMVP) that allows a gene of interest to be studied in the context of an array of promoters, epitope tags, conditional expression modalities, and fluorescent reporters, packaged in 35 custom destination vectors, including adenovirus, lentivirus, PiggyBac transposon, and Sleeping Beauty transposon, in aggregate >108,000 vector permutations. We also used pMVP to build an epigenetic engineering platform, pMAGIC, that packages multiple gRNAs and either Sa-dCas9 or x-dCas9(3.7) fused to one of five epigenetic modifiers. Importantly, via its compatibility with adenoviral vectors, pMAGIC uniquely enables use of dCas9/LSD1 fusions to interrogate enhancers within primary cells. To demonstrate this, we used pMAGIC to target Sa-dCas9/LSD1 and modify the epigenetic status of a conserved enhancer, resulting in altered expression of the homeobox transcription factor PDX1 and its target genes in pancreatic islets and insulinoma cells. In sum, the pMVP and pMAGIC systems empower researchers to rapidly generate purpose-built, customized vectors for manipulation of gene expression, including via targeted epigenetic modification of regulatory elements in a broad range of disease-relevant cell types.
Assuntos
Sistemas CRISPR-Cas/genética , Engenharia Genética/métodos , Vetores Genéticos/genética , Proteínas de Homeodomínio/genética , Transativadores/genética , Transgenes/genética , Adenoviridae/genética , Animais , Elementos de DNA Transponíveis/genética , Elementos Facilitadores Genéticos/genética , Epigenômica/métodos , Edição de Genes/métodos , Regulação da Expressão Gênica/genética , Células HEK293 , Histona Desmetilases/genética , Humanos , Insulinoma/metabolismo , Ilhotas Pancreáticas/metabolismo , Lentivirus/genética , Camundongos , Regiões Promotoras Genéticas/genética , RNA Guia de Cinetoplastídeos/genética , RatosRESUMO
Maternal glucose levels during pregnancy impact the developing fetus, affecting metabolic health both early and later on in life. Both genetic and environmental factors influence maternal metabolism, but little is known about the genetic mechanisms that alter glucose metabolism during pregnancy. Here, we report that haplotypes previously associated with gestational hyperglycaemia in the third trimester disrupt regulatory element activity and reduce expression of the nearby HKDC1 gene. We further find that experimentally reducing or increasing HKDC1 expression reduces or increases hexokinase activity, respectively, in multiple cellular models; in addition, purified HKDC1 protein has hexokinase activity in vitro. Together, these results suggest a novel mechanism of gestational glucose regulation in which the effects of genetic variants in multiple regulatory elements alter glucose homeostasis by coordinately reducing expression of the novel hexokinase HKDC1.