Moss Methods – The Bezanilla Lab

Our Moss Manual

The moss Physcomitrella patens is a powerful model system to study plant gene function. Unique among plants, moss performs efficient homologous recombination enabling precise genetic manipulation, including gene deletions and allele replacements. With a completely sequenced genome, facile genetic alteration, and a relatively simple developmental pattern, moss provides an unparalleled tool for dissecting plant gene function. Moss is simple to propagate and transform, and requires little space to maintain. This lab manual contains information about propagation, transformation, and DNA and protein isolation.

Moss Manual
(Updated: 2012)

Moss Propagation and Culture

Moss should be propagated weekly. A detailed explanation can be found in the moss manual. Watch the video for a brief summary on moss propagation.

Propagation Video

Protoplasting

By removing the cell walls from moss tissue, it is possible to generate a suspension of single cells called protoplasts. Moss protoplasts can be transformed with DNA and then regenerated into whole plants. For details on this procedure, please refer to the moss manual.

Common Assays

Click on an assay to learn more.

Growth Assay

Moss protoplasts given the proper growth conditions will regenerate into a whole plant within a few days. Thousands of plants can be regenerated. Taking pictures of the plants, it is possible to get measurements of overall plant size and shape.

RNAi Assay

Moss performs RNAi-induced gene silencing. Double stranded RNA can be generated by transformation of DNA constructs containing inverted repeats of the sequence targeted for gene silencing. Our laboratory uses a robust RNAi assay that enables rapid identification of gene function.

Complementation Assay

Complementation studies provide validation of loss-of-function studies and mechanistic insights into gene function. By targeting untranslated regions of sequence, RNAi-induced phenotypes can be rescued by expression of a construct containing only the open reading frame.

Available Reagents: Plasmids

Transient and/or Stable Overexpression Vectors

Our Efficient CRISPR-Cas9 Vector System

^NEW!

RNAi Vectors

Two-Fragment Recombination Vectors

Three-Fragment Recombination Vectors

Four-Fragment Recombination Vectors

Available Reagents: Moss Lines

NLS4 A kanamycin-resistant line expressing GFP:GUS localized to the nucleus.
Golgi A hygromycin-resistant line expressing YFP targeted to the Golgi.
Vacuole A kanamycin-resistant line expressing Vam3-mEGFP (mEGFP targeted to the tonoplast membrane).
ER A hygromycin-resistant line expressing mEGFP targeted to the endoplasmic reticulum.
Microtubules A kanamycin-resistant line expressing mEGFP-tubulin.
Actin A hygromycin-resistant line expressing Lifeact-mEGFP.
Peroxisome A hygromycin-resistant line expressing mEGFP targeted to the peroxisome.