11.4 Conditional Deletion


11.4.1 Conditional Knock-out (KO)

Minoru Kubo



It is a powerful tool for characterization of the gene function to analyze a “loss-of-function” mutant with the deficient gene. Although gene KO (= disruptant) in P. patens has been useful for loss-of-function analyses, it is important to avoid lethality of gene KO because the moss is haploid in most of its life cycle. Thus, we developed a conditional KO system by using Cre-loxP system, in which Cre recombinase is expressed under the control of HSP facilitating excision of target gene flanked by loxP sites (Fig.1).



Fig.1 A scheme of conditional KO. HSP: heat shock promoter, Cre: Cre recombinase, YFP: yellow fluorescent protein (Citrine), NLS-mRFP: monomeric red fluorescent protein with nuclear localizing signal.



Materials and methods


How to make a construct

First, we established the parental line harboring Cre recombinase driven by HSP in P. patens genome (HSP::Cre line). To make conditional KO construct for the target gene, of which three genomic regions, 5’UTR (promoter), CDS (gene) and 3’UTR are amplified with proof-reading DNA polymerase from P. patens genomic DNA and directionally inserted at BstZ17I, EcoRV and SmaI in pCKO2, respectively (Fig.2).


Transformation and selection of candidate lines

The fragment integrated to P. patens genome is excised by appropriate restriction enzyme (generally PmeI). The fragments are transformed to HSP::Cre line by PEG-mediated transformation (see Chapter 9.1). For detail of transformation and selection of candidate lines, see Chapter 11.3.


How to induce conditional KO

Transgenic P. patens harboring constructs of conditional KO and HSP::Cre are cultured at 25. To treat heat-shock, they are transferred to 38 for 1hr and then returned to 25. To effectively induce KO, you should carry out this treatment per day twice.


Fig.2 Vector map of pCKO2.

pCKO2 containing loxP sites for excision by Cre recombinase,  yellow fluorescent protein (Citrine) to fuse to CDS for detection of subcellular localization, modified NPTII cassette for transformant selection by G418 and monomeric red florescent protein with nuclear localization signal (NLS-mRFP) for detection of KO cell. Unfortunately, NLS-mRFP is not functional. Cut by BstZ17I, EcoRV and SmaI, blunt ends for ligation of PCR products are generated.

11.4.2 Deletion of a marker gene

Yoshikatsu Sato



In this section, I introduce the method for the deletion of selection marker gene. The pTN182 (G418 resistant cassette), pTN186 (hygromycin resistant cassette), p35S-zeo (Zeocin resistant cassette), and pCtrnNPTII2 (G418 resistant cassette for C-terminal YFP fusion) contain the loxP sites in order to delete the selection marker gene if needed such as 1) poverty of selection marker cassette for generating multiple disruptant and 2) restoring the position of 3'UTR of the target gene.



1. Circular plasmid of pTN75 (Cre recombnase expression vector + hygromycin resistant cassette) is transiently expressed by usual PEG transformation method.

2. Cultivate for ~2 weeks on the hygromycin medium.

3. Inoculate 24 colonies on the BCDAT medium and cultivate for 2 weeks.

4. (Optional) Confirm the loss of resistance. Cultivate for 2 weeks on the drug to which the transformed strain showed tolerant.

5. Confirm deletion of selection marker cassette by Green-PCR

6. Confirm that the candidate strains are hygromycin sensitive.


Key point

Circular plasmid of pTN75 should be used so that efficient expression of Cre recombinase is induced transiently.

テキスト ボックス:

Pact: rice actin promoter

Cre: Cre recombinase gene

TrbcS: rbcS terminator

Pcmv: CaMV 35S promoter

aph4: hygromycin resistant gene

Tcmv: CaMV 35S terminator