Efficient gene trap screening for novel developmental genes using IRES beta geo vector and in vitro preselection.
We have used different gene trap vectors and in vitro preselection of embryonic stem (ES) cells for a large scale screening of insertional mutations in developmentally regulated genes. A gene trap vector was constructed, which contains an internal ribosome entry site (IRES) upstream from a betageo selectable-reporter fusion gene. Analysis of 801 independent integrations revealed that the IRESbetageo vector allows for a global enrichment of about 15 folds in the number of detectable gene trap events when compared with a conventional betageo vector. Characterization of in vitro and in vivo lacZ expression suggested that this IRES-based vector is able to capture a wide range of genes expressed in a variety of tissues and developmental stages, and it can also allow trapping of genes expressed at very low levels in ES cells. A preselection protocol was devised, where gene-trapped ES cells were grown in the presence of specific growth/differentiation factors such as follistatin, nerve growth factor, and retinoic acid. Several gene trap integrations were found to be either activated or repressed by one of these factors. Characterization of lacZ expression during embryogenesis showed a strong enrichment of restricted patterns in vivo after ES cell preselection. These results suggest that a combination of IRESbetageo vector and in vitro preselection is more effective for the capture and mutation of a large number of developmental genes.
Bonaldo
P
P
Department of Molecular Cell Biology, Max Planck Institute of Biophysical Chemistry, Göttingen, 37077, Germany.
Chowdhury
K
K
Stoykova
A
A
Torres
M
M
Gruss
P
P
D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D018507:Gene Expression Regulation, Developmental; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000235:genetics*; D007763:Lac Operon; D051379:Mice; D008822:Mice, Transgenic; D012270:Ribosomes / Q000235:genetics*; D013234:Stem Cells