PHYSCOmanualver. 1.4

Yuji Hiwatashi, Tomomichi Fujita, Yoshikatsu Sato,

Takako Tanahashi, Tomoaki Nishiyama, Keiko Sakakibara,

Rumiko Kofuji, Naoki Aono, Naomi Sumikawa,

Takashi Murata, and Mitsuyasu Hasebe

National Institute for Basic Biology, Okazaki, Japan

up date: 4 Jan. 2008

16. Available full-length cDNA is updated (12/13, 2007)

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

2.1 Germination of spores

2.2 Culture and storage of protonemata and gametophores

2.3 Induction of antheridia, archegonia 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

3. Observation

3.1 Observation of protonemata and gametophores

3.2 Observation of protonema colony morphology

3.3 Observation of mitosis in a protonemal cell

3.4 Light response

3.5 Gravitropic response on caulonemata

3.6 Observation of antheridia and archegonia

3.7 Observation of Sperms

3.8 Observation of archegonia and embryos by confocal laser scanning microscopy

3.9 DAPI, Hoechst33342, and PI Staining of protonemata

3.10 Visualization of cell wall with calcofluor

3.11 GUS staining

3.12 Microtubule by indirect immunofluorescence microscopy

3.13 Live imaging

3.14 Light and electron microscopy of archegonia embedded with epoxy resin

4. Gene isolation

4.1 Genomic DNA extraction

4.2 Simple DNA extraction for PCR screening

4.3 Isolation of genomic fragment by TAIL-PCR

4.4 RNA Extraction

4.5 RACE

4.6 Library Screening

4.7 Miniprotoplast preparation for BAC library

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 development

7.1 Hormone treatments

7.2 Cytoskeletal inhibitors

8. Flow Cytometry Analysis

9. Transformation

9.1 PEG-mediated transformation

9.2 Transient expression of a foreign gene using particle bombardment

10. Expression analyses

10.0 Retrieving Genomic Sequences for Gene Targeting Using the Available Web-Based Browsers

10.1 Insertion of GUS (GFP, YFP) genes to form a fusion protein with a target gene

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

11. Loss of function analyses

11.1 Gene disruption

11.2 RNA interference

12. Over and ectopic exression

12.1 Constitutive overexpression of cDNA or cDNA with tags

12.2 Transient overexpression

13. PHYSCObase

14. Physcomitrella genome project

16. Available full length cDNA

17. Course and Workshop