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Generation of LAMC2-EGFP knock-in human pancreatic cancer cells

ABSTRACT: Generation of LAMC2-EGFP knock-in human pancreatic cancer cells

PROVIDER: PRJEB52044 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR9467520_1.fastq.gz	Fastqsanger.gz
	ERR9467520_2.fastq.gz	Fastqsanger.gz
	ERR9467511_1.fastq.gz	Fastqsanger.gz
	ERR9467511_2.fastq.gz	Fastqsanger.gz
	ERR9467521_1.fastq.gz	Fastqsanger.gz

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Generation of LAMC2-EGFP knock-in human pancreatic cancer cells

Project description:Since LAMC2 is a secreted molecule present in the extracellular matrix of the cells, was designed a strategy based on CRISPR/Cas9-mediated homologous recombination to mark LAMC2 cells in human PDACs. A Cas9 single-guide RNAs complementary to sequences overlapping the stop codon of the LAMC2 locus was designed and a donor vector that contained LAMC2 homology arms flanking an EGFP reporter cassette positioned immediately upstream of the stop codon was generated. LF2A self-cleavage peptide in frame with EGFP so that LAMC2-EGFP locus was expressed as a single mRNA was added, whereas the resulting polypeptide was cleaved in the two encoded proteins, LAMC2 and EGFP. L3.6pl and PANC-1 cells were nucleofected with the donor vector together with a guide-RNA-Cas9 (guide). The engineered cells were subcutaneously injected in CD1 male mice and RNA-seq was performed on LAMC2-EGFP+ and EGFP- -derived tumors.

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ZHBTc4 ES cells stably expressing EGFP vs Nr5a2-EGFP or Oct4-EGFP vs Nr5a2-EGFP

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