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Gene targeting using a promoterless gene trap vector ("targeted trapping") is an efficient method to mutate a large fraction of genes.

ABSTRACT: A powerful tool for postgenomic analysis of mammalian gene function is gene targeting in mouse ES cells. We report that homologous recombination using a promoterless gene trap vector ("targeting trapping") yields targeting frequencies averaging above 50%, a significant increase compared with current approaches. These high frequencies appear to be due to the stringency of selection with promoterless constructs, because most random insertions are silent and eliminated by drug selection. The promoterless design requires that the targeted gene be expressed in ES cells at levels exceeding a certain threshold (which we estimate to be approximately 1% of the transferrin receptor gene expression level, for the secretory trap vector used here). Analysis of 127 genes that had been trapped by random (nontargeted) gene trapping with the same vector shows that virtually all are expressed in ES cells above this threshold, suggesting that targeted and random trapping share similar requirements for expression levels. In a random sampling of 130 genes encoding secretory proteins, about half were expressed above threshold, suggesting that about half of all secretory genes are accessible by either targeted or random gene trapping. The simplicity and high efficiency of the method facilitate systematic targeting of a large fraction of the genome by individual investigators and large-scale consortia alike.

SUBMITTER: Friedel RH

PROVIDER: S-EPMC1193537 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Camera trapping to study wildlife allows for data collection, without the need to capture animals. Traditionally, camera traps have been used to target larger terrestrial mammal species, though recently novel methods and adjustments in procedures have meant camera traps can be used to study small mammals. The selfie trap (a camera trapping method) may present robust sampling and ecological study of small mammals. This study aimed to evaluate the selfie trap method in terms of its ability to detect species and estimate population density. To address this aim, standard small mammal live trapping was undertaken, immediately followed by camera trapping using the selfie trap. Both methods were set to target the arboreal sugar glider (Petaurus breviceps) and semi-arboreal brown antechinus (Antechinus stuartii). The more ground-dwelling bush rat (Rattus fuscipes) was also live trapped and recorded on camera. Across four survey areas, the probability of detection for each of the three species was higher for selfie traps than for live trapping. Spatially explicit capture-recapture models showed that selfie traps were superior at estimating density for brown antechinus and sugar gliders, when compared to simulated live trapping data. Hit rates (number of videos per various time intervals) were correlated with abundance. When correlating various hit rate intervals with abundance, the use of 10-min hit rate was best for predicting sugar glider abundance (R2 = 0.94). The abundance of brown antechinus was estimated from selfie traps using a 24-h hit rate as a predictor (R2 = 0.85). For sugar gliders, the selfie trap can replace live trapping as individuals can be identified through their unique facial stripes and natural ear scars, and thus used in capture-recapture analysis. This method may be useful for monitoring the abundance of other small mammal species that can also be individually recognized from photographs.Supplementary informationThe online version contains supplementary material available at 10.1007/s13364-022-00643-5.

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Gene targeting using a promoterless gene trap vector ("targeted trapping") is an efficient method to mutate a large fraction of genes.

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