Rapid and Efficient Genetic Manipulation of Intestinal Organoids Under Transient Two-Dimensional Culture Conditions

Genetic manipulation of cells represents one of the fundamental techniques in current biological research, essential for elucidating the regulatory mechanisms underlying various biological phenomena, modeling many diseases, and investigating their therapeutic interventions. However, in the context of organoid biology, the manipulation of target genes and the selection of resultant genetically modified clones often encounter significant technical challenges due to the typically low gene introduction efficiency. This difficulty is largely attributed to the three-dimensional culture of organoids within the basement membrane matrix, which can be substantially mitigated by employing transient two-dimensional culture conditions during the introduction of genes. This section describes a method involving the temporary use of two-dimensional culture of intestinal organoids on collagen gel, followed by gene introduction using lipofection or lentiviral vectors. This method allows rapid and efficient genetic manipulation of intestinal organoids and can be applied to organoids from other organs.