Among the insertional mutagenesis techniques used in the current international knockout mouse project (KOMP) on the inactivation of all mouse genes in embryonic stem (ES) cells, random gene trapping has been playing a major role. Gene-targeting experiments have also been performed to individually and conditionally knockout the remaining ‘difficult-to-trap’ genes. Here, we show that transcriptionally silent genes in ES cells are severely underrepresented among the randomly trapped genes in KOMP. Our conditional poly(A)-trapping vector with a common retroviral backbone also has a strong bias to be integrated into constitutively transcribed genome loci. Most importantly, conditional gene disruption could not be successfully accomplished by using the retrovirus vector because of the frequent development of intra-vector deletions/rearrangements. We found that one of the cut and paste-type DNA transposons, Tol2 , can serve as an ideal platform for gene-trap vectors that ensures identification and conditional disruption of a broad spectrum of genes in ES cells. We also solved a long-standing problem associated with multiple vector integration into the genome of a single cell by incorporating a mixture of differentially tagged Tol2 transposons. We believe our strategy indicates a straightforward approach to mass-production of conditionally disrupted alleles for genes in the target cells.

Summary Random gene trapping is the application of insertional mutagenesis techniques that are conventionally used to inactivate protein‐coding genes in mouse embryonic stem (ES) cells. Transcriptionally silent genes are not effectively targeted by conventional random gene trapping techniques, thus we herein developed an unbiased poly (A) trap (UPATrap) method using a Tol2 transposon, which preferentially integrated into active genes rather than silent genes in ES cells. To achieve efficient trapping at transcriptionally silent genes using random insertional mutagenesis in ES cells, we generated a new diphtheria toxin (DT)‐mediated trapping vector, DTrap that removed cells, through the expression of DT that was induced by the promoter activity of the trapped genes, and selected trapped clones using the neomycin‐resistance gene of the vector. We found that a double‐DT, the dDT vector, dominantly induced the disruption of silent genes, but not active genes, and showed more stable integration in ES cells than the UPATrap vector. The dDT vector disrupted differentiated cell lineage genes, which were silent in ES cells, and labeled trapped clone cells by the expression of EGFP upon differentiation. Thus, the dDT vector provides a systematic approach to disrupt silent genes and examine the cellular functions of trapped genes in the differentiation of target cells and development.

‘Situ’ Lotus and ‘Situ’ Cibuntu are small ponds located in urban areas at Cibinong Science Center-Botanical Garden, National Research and Innovation Agency, Indonesia. This study was conducted to determine the development of the introduced freshwater shrimp Macrobrachium lanchesteri in that ponds. Observations were carried out in January and February 2019. There are four sampling points in each pond. The number of shrimp obtained at Lotus Pond is more than Cibuntu Pond. The average total length and weight of shrimp obtained at Lotus Pond during the first observation were 1.61 – 2.55 cm and 0.07 – 0.20 gram, while the second was 1.67 – 2.27 cm and 0.11 – 0.31 gram. Meanwhile, M. lanchesteri obtained at Cibuntu Pond in the first observation had an average total length of 2.20 – 2.63 cm and weight 0.16 – 0.20 gram; and when the second sampling was 2.27 – 3.00 cm and 0.16 – 0.34 gram. Proximate analysis of shrimp from Cibuntu Pond showed that this shrimp’s protein, fat, ash, crude fiber, and carbohydrate content were 58.68%; 10.70%; 15.31%; 7.70%, and 7.61%, respectively. The water quality observed in both ponds is still good and can support the shrimp’s life. Utilization of this shrimp as an alternative source of protein can be done if pond water quality is always maintained in the future.