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By coupling the pseudotyped virus with transgene expression of the G-protein and the <jats:italic>avian leukosis and sarcoma virus subgroup A receptor<\/jats:italic> (TVA), which is necessary for cell entry of the virus, researchers can investigate specific neuronal populations. Responder mouse lines, like the R\u03a6GT mouse line, carry the genes encoding the G-protein and TVA under Cre-dependent expression. These mouse lines are valuable tools because they reduce the number of viral injections needed compared to when using helper viruses. Since R\u03a6GT mice do not express Cre themselves, introducing the pseudotyped rabies virus into their brain should not result in viral cell entry or spread.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>We present a straightforward flowchart for adequate controls in tracing experiments, which we employed to demonstrate Cre-independent expression of TVA in R\u03a6GT mice.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>Our observations revealed TVA leakage, indicating that R\u03a6GT mice should be used with caution for transgene expression of TVA. Inaccurate tracing outcomes may occur if TVA is expressed in the absence of Cre since background leakage leads to nonspecific cell entry. Moreover, conducting appropriate control experiments can identify the source of potential caveats in virus-based neuronal tracing experiments.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12868-024-00848-1","type":"journal-article","created":{"date-parts":[[2024,2,21]],"date-time":"2024-02-21T14:02:52Z","timestamp":1708524172000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A flowchart for adequate controls in virus-based monosynaptic tracing experiments identified Cre-independent leakage of the TVA receptor in R\u03a6GT mice"],"prefix":"10.1186","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4604-2949","authenticated-orcid":false,"given":"Anna","family":"Velica","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6418-5460","authenticated-orcid":false,"given":"Klas","family":"Kullander","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2024,2,21]]},"reference":[{"issue":"5","key":"848_CR1","first-page":"639","volume":"53","author":"IR Wickersham","year":"2007","unstructured":"Wickersham IR, Lyon DC, Barnard RJO, Mori T, Finke S, Conzelmann KK, et al. 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