Guillaume Burnet, Chun-Wei Allen Feng, Ka Man Fiona Cheung, Josephine Bowles, Cassy M. Spiller
{"title":"Generation and characterization of a Ddx4-iCre transgenic line for deletion in the germline beginning at genital ridge colonization","authors":"Guillaume Burnet, Chun-Wei Allen Feng, Ka Man Fiona Cheung, Josephine Bowles, Cassy M. Spiller","doi":"10.1002/dvg.23511","DOIUrl":null,"url":null,"abstract":"<p>Germline-specific Cre lines are useful for analyses of primordial germ cell, spermatogonial and oogonial development, but also for whole-body deletions when transmitted through subsequent generations. Several germ cell specific Cre mouse strains exist, with various degrees of specificity, efficiency, and temporal activation. Here, we describe the CRISPR/Cas9 targeted insertion of an improved <i>Cre</i> (<i>iCre</i>) sequence in-frame at the 3′ end of the <i>Ddx4</i> locus to generate the <i>Ddx4-P2A-iCre</i> allele. Our functional assessment of this new allele, designated <i>Ddx4</i><sup><i>iCreJoBo</i></sup>, reveals that Cre activity begins in PGCs from at least E10.5, and that it achieves higher efficiency for early gonadal (E10.5–12.5) germline deletion when compared to the inducible <i>Oct4</i><sup><i>CreERT2</i></sup> line. We found the <i>Ddx4</i><sup><i>iCreJoBo</i></sup> allele to be hypomorphic for <i>Ddx4</i> expression and homozygous males, but not females, were infertile. Using two reporter lines (<i>R26R</i><sup><i>LacZ</i></sup> and <i>R26R</i><sup><i>tdTomato</i></sup>) and a floxed gene of interest (<i>Cripto</i><sup><i>flox</i></sup>) we found ectopic activity in multiple organs; global recombination (a common feature of germline Cre alleles) varies from 10 to 100%, depending on the particular floxed allele. There is a strong maternal effect, and therefore it is preferable for <i>Ddx4</i><sup><i>iCreJoBo</i></sup> to be inherited from the male parent if ubiquitous deletion is not desired. With these limitations considered, we describe the <i>Ddx4</i><sup><i>iCreJoBo</i></sup> line as useful for germline studies in which early gonadal deletion is required.</p>","PeriodicalId":12718,"journal":{"name":"genesis","volume":"61 1-2","pages":""},"PeriodicalIF":2.4000,"publicationDate":"2023-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/dvg.23511","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"genesis","FirstCategoryId":"99","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/dvg.23511","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"DEVELOPMENTAL BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Germline-specific Cre lines are useful for analyses of primordial germ cell, spermatogonial and oogonial development, but also for whole-body deletions when transmitted through subsequent generations. Several germ cell specific Cre mouse strains exist, with various degrees of specificity, efficiency, and temporal activation. Here, we describe the CRISPR/Cas9 targeted insertion of an improved Cre (iCre) sequence in-frame at the 3′ end of the Ddx4 locus to generate the Ddx4-P2A-iCre allele. Our functional assessment of this new allele, designated Ddx4iCreJoBo, reveals that Cre activity begins in PGCs from at least E10.5, and that it achieves higher efficiency for early gonadal (E10.5–12.5) germline deletion when compared to the inducible Oct4CreERT2 line. We found the Ddx4iCreJoBo allele to be hypomorphic for Ddx4 expression and homozygous males, but not females, were infertile. Using two reporter lines (R26RLacZ and R26RtdTomato) and a floxed gene of interest (Criptoflox) we found ectopic activity in multiple organs; global recombination (a common feature of germline Cre alleles) varies from 10 to 100%, depending on the particular floxed allele. There is a strong maternal effect, and therefore it is preferable for Ddx4iCreJoBo to be inherited from the male parent if ubiquitous deletion is not desired. With these limitations considered, we describe the Ddx4iCreJoBo line as useful for germline studies in which early gonadal deletion is required.
期刊介绍:
As of January 2000, Developmental Genetics was renamed and relaunched as genesis: The Journal of Genetics and Development, with a new scope and Editorial Board. The journal focuses on work that addresses the genetics of development and the fundamental mechanisms of embryological processes in animals and plants. With increased awareness of the interplay between genetics and evolutionary change, particularly during developmental processes, we encourage submission of manuscripts from all ecological niches. The expanded numbers of genomes for which sequencing is being completed will facilitate genetic and genomic examination of developmental issues, even if the model system does not fit the “classical genetic” mold. Therefore, we encourage submission of manuscripts from all species. Other areas of particular interest include: 1) the roles of epigenetics, microRNAs and environment on developmental processes; 2) genome-wide studies; 3) novel imaging techniques for the study of gene expression and cellular function; 4) comparative genetics and genomics and 5) animal models of human genetic and developmental disorders.
genesis presents reviews, full research articles, short research letters, and state-of-the-art technology reports that promote an understanding of the function of genes and the roles they play in complex developmental processes.