The aim of this practical course is to familiarize students with microinjection and micromanipulation techniques on the model of round worm (C. elegans) and clawed frogs (Xenopus laevis and Xenopus tropicalis). Students will have also the oportunity to work with advanced microscopic systems such as light sheet and live cell immaging microscopy.
Last update: Krylov Vladimír, doc. RNDr. Ing., Ph.D. (18.09.2019)
Cílem praktik je seznámit studenty s mikroinjekčními a mikromanipulačními technikami na modelu háďátka (C. elegans) a drápatky (X. laevis a X. tropicalis). V kurzu bude taktéž využita light sheet a live cell imaging mikroskopie.
Last update: Krylov Vladimír, doc. RNDr. Ing., Ph.D. (18.09.2019)
Literature -
Literature will be available at each course.
Last update: Šebková Nataša, RNDr., Ph.D. (24.10.2019)
Literatura bude k dispozici na každém kurzu.
Last update: Šebková Nataša, RNDr., Ph.D. (24.10.2019)
Requirements to the exam -
course attendance, working out the practical course protocols, to be able to communicate in English language.
Last update: Krylov Vladimír, doc. RNDr. Ing., Ph.D. (26.08.2019)
docházka, vypracování protokolů z cvičení, zvládnout výuku v anglickém jazyce.
Last update: Šebková Nataša, RNDr., Ph.D. (21.02.2018)
Syllabus -
Task 1: RNA interference of the embryonic lethal gene in C. elegans.
- C. elegans morphology, principle of the RNA interference
- in vitro synthesis of dsRNA
- preparation of microinjection tools (capillary, pads for the C. elegans immobilization)
- familiarization with the micromanipulator, microinjection process
Task 2: Study of migration potential of stem cells
- what are stem cells?
- meet Xenopus!
- preparation of RFP positive Sertoli stem cells derived from the testes of X. tropicalis juvenile male for the microinjection
- preparation of media, capillary and microinjection tools
- microinjection of Sertoli stem cells into X. tropicalis tadpoles
- light sheet microscopy - observation of migration routes of microinjected cells in living tadpoles
- preparation of photodocumentation
The course is taught with the support of the project reg. number CZ.02.2.69/0.0/0.0/16_015/0002362
Last update: Krylov Vladimír, doc. RNDr. Ing., Ph.D. (18.09.2019)
Úloha1: RNA interference embryonálně letálního genu u C. elegans
- seznámení s morfologií C. elegans, princip RNA interference
- in vitro syntéza dsRNA
- příprava nástrojů pro mikroinjekci (kapiláry, podložky pro imobilizaci C. elgens
- seznámení s mikromanipulátorem, vlastní mikroinjekce
Úloha2: Studium migračního potenciálu kmenových buněk
- problematika kmenových buněk
- seznámení se s modelovým organizmem Xenopus tropicalis
- příprava RFP pozitivních Sertoliho kmenových buněk odvozených z varlat X. tropicalis a pěstovaných v buněčné kultuře pro mikroinjekční experiment
- analýza buněk před mikroinjekcí pomocí cell counteru
- příprava médií, kapilár a nástrojů pro mikroinjekci
- mikroinjekce Sertoliho kmenových buněk do pulců X. tropicalis
- light sheet mikroskopie - pozorování a záznam migračních tras mikroinjikovaných buněk
- příprava fotodokumentace.
Předmět je vyučován za podpory projektu Zvýšení kvality vzdělávání na UK a jeho relevance pro potřeby trhu práce, reg.č. CZ.02.2.69/0.0/0.0/16_015/0002362.
Last update: Krylov Vladimír, doc. RNDr. Ing., Ph.D. (18.09.2019)
Learning outcomes -
After successful completion of the course, the student will be able to:
1. Remember and understand
Describe the principles of RNA interference, the basic morphology of Caenorhabditis elegans, and the biological characteristics of the model organisms Xenopus laevis and Xenopus tropicalis.
Explain the principles, advantages, and limitations of microinjection and micromanipulation techniques, including the basic concepts of light sheet and live cell imaging microscopy.
2. Apply
Prepare and handle microinjection tools, including pulling and testing glass capillaries, preparing immobilization pads for C. elegans, and correctly setting up and operating a micromanipulator and microinjector.
Perform in vitro synthesis of dsRNA for an embryonically lethal gene of C. elegans and apply it by microinjection to induce RNA interference with a documented observable phenotype.
Prepare RFP‑positive cells derived from X. tropicalis testes for microinjection, including maintenance in cell culture, cell dissociation and cell counting using a cell counter.
3. Analyze
Analyze and interpret the success of RNAi and microinjection experiments in C. elegans (e.g. survival, penetrance of phenotype) and identify probable technical or conceptual sources of errors.
Evaluate and interpret migration patterns of microinjected RFP-positive cells in X. tropicalis tadpoles using light sheet microscopy recordings, including basic quantitative description of migration routes.
4. Create
Design a simple experimental workflow using microinjection or micromanipulation to address a defined question in gene function or cell migration in one of the course model organisms, including appropriate controls.
Produce a complete experimental record, including an English‑language lab protocol, parameter settings, and photographic documentation suitable for inclusion in a research‑style laboratory report.
5. Evaluate
Critically assess the quality and reproducibility of their own microinjection and imaging data and propose specific modifications to improve experimental design or technical execution in future experiments.
Communicate and defend their experimental approach, results, and limitations in spoken and written English using appropriate terminology from molecular, developmental, and cell biology.
SMART alignment
Specific: Each outcome refers to clearly defined experimental techniques, model organisms, and types of data (e.g. RNAi in C. elegans, light sheet imaging of cell migration).
Measurable: Achievement is assessed through attendance, evaluated lab protocols, quality of experimental results, and oral/written communication in English.
Achievable: Outcomes correspond to skills typically attained in laboratory‑intensive molecular biology and developmental biology courses at bachelor/early master level.
Relevant: Outcomes develop practical and analytical competencies in RNAi, cell biology, and advanced microscopy that are directly relevant for modern biomedical research.
Time‑bound: All outcomes are to be demonstrated within the duration of the course through continuous assessment of exercises and final lab records.
Last update: Tlapáková Tereza, RNDr., Ph.D. (29.01.2026)
Po úspěšném absolvování kurzu bude student schopen:
1. Zapamatování a porozumění
Popsat principy RNA interference, základní morfologii C. elegans a biologii modelových organizmů Xenopus laevis a X. tropicalis.
Vysvětlit principy a možnosti použití mikroinjekčních a mikromanipulačních technik, včetně základů light sheet a live cell imaging mikroskopie.
