PHYSCOmanualver. 2.0

National Institute for Basic Biology, Okazaki, Japan

Up date: 24 July 2012


This manual was updated from ver. 1.4 to ver. 2.0. Link to ver. 1.4.

We found that 7133 promoter we have been used has a sequence of 7113 promoter. Please read all “7133” in this manual as “7113”.

0. Preface

00. Selected review articles

1. Strains and life cycle

2. Cultivation of Physcomitrella patens

2.1 Spore germination

2.2 Culture and storage of protonemata and gametophores

2.3 Induction of gametangia and sporophytes

2.4 Sporangium collection and storage

2.5 Protonemata cultivation between two thin layers of agar-gelatin in liquid medium for high quality microscope observation

2.6 Crosses of mutants defective in the sporophyte formation

2.7 Culture in a thin layer of agar medium spread in a glass-bottom dish

2.8 Removal of contaminants

3. How to Observe Physcomitrella patens

3.1 Observation of protonemata and gametophores

3.2 Observation of antheridia and archegonia

3.3 Observation of Sperms

3.4 Observation of archegonia and embryos by confocal laser scanning microscopy

3.5 DAPI, Hoechst33342, and PI Staining of protonemata

3.6 Visualization of cell walls with calcofluor

3.7 GUS staining

3.8 Observation of microtubules with indirect immunofluorescence microscopy

3.9 Live imaging

3.10 Light and electron microscopy of protonemata embedded with epoxy resin

4. Gene isolation

4.1 Genomic DNA extraction

4.2 Green PCR

4.3 Isolation of genomic fragment by TAIL-PCR

4.4 RNA Extraction

4.5 RACE

4.6 Library Screening

5. DNA and RNA gel-blot and RT-PCR analyses

5.1 DNA gel-blot analysis

5.2 RT-PCR

5.3 RNA gel-blot analysis

6. Western blotting

7. Effects of drugs on moss development

7.1 Hormone treatments

7.2 Cytoskeletal inhibitors

7.3 Cell cycle inhibitors

8. Flow Cytometry Analysis

9. How to transform Physcomitrella patens

9.1 PEG-mediated transformation

9.2 Transient expression of a foreign gene using particle bombardment

9.3 Transient overexpression

9.4 Agrobacterium-mediated transformation

10. Cellular localization of a protein fused with a fluorescent protein (GFP, YFP, RFP)

10.1 Expression and localization of a fusion protein of a targeted gene and a reporter gene

10.2 How to observe the cellular localization of a fluorescent protein

11. Loss/Gain of Function Analyses

11.1 Gene deletion

11.2 RNA interference

11.3 Conditional knock down for specific gene by artificial microRNA (amiRNA)

11.4 Conditional Deletion

11.5 Dominant repression by chimeric repressor silencing technology (CRES-T)

11.6 Overexpression

11.7 Inducible expression

11.8 Chromatin Immunoprecipitation

12. How to analyze the phenotype of a mutant

12.1 Observation of protonema colony morphology

12.2 Protonema development

12.3 Light response

12.4 Gravitropic response on caulonemata

12.5 Regeneration of protonemata from excised leaves

13. How to use genome information

13.1 Searching homologues of your protein coding gene

14. Analyses by SOLiD (DGE, ChIP-seq, Small RNA-seq)

15. Forward Genetic Approach: Deletion Mutants