ABSTRACT
The combination of genome-editing and epitope tagging provides a powerful strategy to study proteins with high affinity and specificity while preserving their physiological expression patterns. However, stably modifying endogenous genes in cells that do not allow for clonal selection has been challenging. Here, we present a simple and fast strategy to generate stable, endogenously tagged alleles in a non-transformed cell culture model. At the example of piwi in Drosophila ovarian somatic sheath cells, we show that this strategy enables the generation of an N-terminally tagged protein that emulates the expression level and subcellular localization of the wild type protein and forms functional Piwi-piRNA complexes. We further present a concise workflow to establish endogenously N-terminally and C-terminally tagged proteins, and knockout alleles through rapid selection of cell pools in fly and human models.
Subject(s)
Drosophila Proteins , Drosophila , Animals , Drosophila/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Female , Gene Editing , Genes, Reporter , Humans , Ovary/metabolism , RNA, Small Interfering/metabolismABSTRACT
Sexual and reproductive health (SRH) services are a necessity for marginalized persons such as the displaced. The protocol describes an intervention that can contribute to overcoming challenges associated with SRH service delivery of three selected reproductive health (RH) services: HIV/AIDS, contraception, and cervical cancer screening. A pre-and post-intervention approach will be used to evaluate the effect of an intervention with trained Community-Based Reproductive Health Personnel (CBRHP) and/or mHealth technology within the selected IDP camps. Three (3) months of health education through the CBRHP and/or via mHealth technology will be provided. Using a questionnaire, interviews, and Focus Group Discussion (FGD) guide, the researcher will assess the suitability of this intervention to attain the objectives. Data analysis will be done with SPSS version 26. Univariate analysis will include mean and standard deviation, bivariate analysis will include a chi-square test of goodness for the association of variables, and McNemer's test to evaluate the effect of the intervention by comparing consistency in response across the variables under study. Multivariate analysis will be used to assess if sociodemographic, knowledge and health service impacts access and use of RH services. For qualitative analysis, findings will be grouped into themes. The outcomes of each theme will be used to complement the findings of the quantitative analysis. The primary outcome measures will include the level of knowledge of SRH, the number of people who want to access RH services and which RH services are most sought by the respondents. If the use of CBRHP is successful, there will be an increase in knowledge and use of HIV/AIDS, contraception and cervical cancer services. Challenges associated with access and uptake of RH services will also be assessed.
Les services de santé sexuelle et reproductive (SSR) sont une nécessité pour les personnes marginalisées telles que les personnes déplacées. Le protocole décrit une intervention qui peut contribuer à surmonter les défis associés à la prestation de services de SSR de trois services de santé reproductive (SR) sélectionnés : VIH/SIDA, contraception et dépistage du cancer du col de l'utérus. Une approche pré- et post-intervention sera utilisée pour évaluer l'effet d'une intervention avec du personnel de santé reproductive à base communautaire (CBRHP) formé et/ou la technologie mHealth au sein des camps de déplacés internes sélectionnés. Trois (3) mois d'éducation sanitaire via le CBRHP et/ou via la technologie mHealth seront dispensés. À l'aide d'un questionnaire, d'entretiens et d'un guide de discussion de groupe (FGD), le chercheur évaluera l'adéquation de cette intervention pour atteindre les objectifs. L'analyse des données sera effectuée avec SPSS version 26. L'analyse univariée inclura la moyenne et l'écart type, l'analyse bivariée comprendra un test de qualité du chi carré pour l'association des variables et le test de McNemer pour évaluer l'effet de l'intervention en comparant la cohérence de réponse pour les variables étudiée. Une analyse multivariée sera utilisée pour évaluer si les services sociodémographiques, les connaissances et les services de santé ont un impact sur l'accès et l'utilisation des services de SR. Pour l'analyse qualitative, les résultats seront regroupés en thèmes. Les résultats de chaque thème seront utilisés pour compléter les résultats de l'analyse quantitative. Les principales mesures des résultats incluront le niveau de connaissances en matière de SSR, le nombre de personnes souhaitant accéder aux services de SR et quels services de SR sont les plus recherchés par les répondants. Si l'utilisation du CBRHP réussit, il y aura une augmentation des connaissances et de l'utilisation des services liés au VIH/SIDA, à la contraception et au cancer du col de l'utérus. Les défis associés à l'accès et à l'utilisation des services de SR seront également évalués.
Subject(s)
Acquired Immunodeficiency Syndrome , Reproductive Health Services , Uterine Cervical Neoplasms , Humans , Female , Reproductive Health/education , Early Detection of Cancer , Uterine Cervical Neoplasms/prevention & control , Sexual Behavior , Health EducationABSTRACT
The harnessing of the CRISPR-Cas9 system allows for quick and inexpensive genome editing in tissue culture models. Traditional CRISPR-Cas9 genome editing techniques rely on the ability of single progenitor cells to expand into new pools in a process known as clonal expansion. This is a significant technical challenge that is difficult to overcome for nontransformed cell culture models such as Drosophila ovarian somatic sheath cells (OSCs). OSCs are a unique ex vivo model for epigenetic regulation by PIWI-interacting RNAs (piRNAs) that establish restriction of mobile genetic elements in germ cells to protect genome integrity. Here, we provide a protocol to generate endogenously tagged proteins and gene knockouts without the need for clonal selection. We combine CRISPR-Cas genome editing and knockin of antibiotic selection markers to generate edited cell pools. At the example of Drosophila piwi in OSCs, we demonstrate a strategy that relies on the insertion of an artificial intron to accommodate a selection marker with minimal disturbance of the resulting mRNA. In brief, our donor cassette contains a peptide tag and an optimized intron that accommodates a selection marker driven by an independent promoter on the other genomic strand. The selection marker is transcribed as an independent mRNA, and the intron is efficiently removed from the mRNA encoding the endogenously tagged (endo-tagged) piwi gene. The endo-tagged Piwi protein is expressed at wild-type levels and appropriately localizes to the nucleus of OSCs. We also describe strategies for C-terminal tagging and generation of knockout alleles in OSCs and in human embryonic kidney cells, discuss different design strategies, and provide a plasmid toolkit (available at Addgene). Our protocol enables robust genome editing in OSCs for the first time and provides a simple and time-saving alternative for other cell culture systems. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Design and cloning of single-guide RNA plasmids Basic Protocol 2: Design and cloning of donor template plasmids for epitope tagging Alternate Protocol: Design and cloning of donor template plasmids for gene knockout Basic Protocol 3: Transfection and selection of edited cell pools.