De novo assembly and delivery to mouse cells of a 101 kb functional human gene.
Genetics
; 218(1)2021 05 17.
Article
em En
| MEDLINE
| ID: mdl-33742653
ABSTRACT
Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here, we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Engenharia Genética
/
Clonagem Molecular
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Genetics
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos