Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors

Abstract

Highlights d Most mouse Cre driver lines tested exhibited variable rates of germline recombination d Germline recombination exhibits parental sex bias and target locus selectivity d Similar principles apply to multiple organisms and recombinase systems d Guidelines are provided for detecting and minimizing unwanted germline recombination

Tsc1 haploinsufficiency in Nkx2.1 cells upregulates hippocampal interneuron mTORC1 activity, impairs pyramidal cell synaptic inhibition, and alters contextual fear discrimination and spatial working memory in mice

“…In experiments with injection of AAV-based constructs (AAV2/9-EF1a-DIO-EYFP [Addgene #27056] and AAV9-EF1a-DIO-hChR2(H134R)-EYFP [Addgene #20298]), heterozygous Nkx2.1 Cre/wt mice served as the control. Although some germline recombination has been observed by others using Nkx2.1 Cre driver mice [20], we did not observe germline recombination in any of our experiments using EYFP reporters (n = 53 Nkx2.1 Cre/wt ;Tsc1 wt/wt mice and 57 Nkx2.1 Cre/wt ;Tsc1 f/wt mice).…”

Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

“…Only cells in intermediate positions, that express sufficiently high Pax6 levels to be able to induce the DA-N phenotype, while at the same time having sufficient Vax1 levels to suppress this differentiation pathway, will show a DA-N phenotype after Vax1 removal. In addition, other factors, like lower recombination efficiency of the Vax1 allele versus the R26tdTomato allele, might further diminish the number of cells in which the impact of gene inactivation can be studied ( Long and Rossi, 2009 ; Luo et al, 2020 ). Thus, while our electroporation approach allows targeting and manipulating specific stem cell compartments, it also has limitations that restrict interpretation.…”

The Villin1 Gene Promoter Drives Cre Recombinase Expression in Extraintestinal Tissues

“…Our findings highlight the necessity of rigorously characterizing transgenic lines in all studies to ensure accurate data interpretation and reproducibility. Just as some Cre lines presumed to be selective for the nervous system were later found to be expressed in the germline or gut epithelium, 9,10 our data show that Cre lines commonly used to study intestinal epithelial biology can be active in the nervous system and other tissues that might affect gut functions. These observations do not detract from the value of these genetic tools, but they do emphasize the need for careful reagent choice, data interpretation, and methods reporting in all studies using these mice.…”

“…Recombination efficiency at different loxP sites and genomic loci can vary. 9 Thus, although our findings provide guidance on Cre activity in these 4 Vil1 transgenic lines, it is possible that this activity may manifest differently in the context of other floxed alleles. Rosa26 Ai9/þ mouse brain shows TdTomato expression in cells within a periventricular region of the forebrain consistent with the anterodorsal thalamic nucleus (A and B) but not in the cerebral cortex (A and C).…”