- Chalberg TW, Portlock JL, Olivares EC,
Thyagarajan B, Kirby PJ, Hillman RT, Hoelters J,
Calos MP. (2006). Integration specificity of
phage φC31 integrase in the human genome. J Mol
Biol. 357(1):28-48. [Abstract]
Applications in Gene
We have developed approaches that
cure hemophilia in disease model mice, by using
hydrodynamic DNA injection and phiC31 integrase
to bring about permanent integration of the
factor VIII or IX genes in the liver:
- Keravala, A.,
Chavez, C.L., Hu, G., Woodard, L.E., Monahan,
P.E., and Calos, M.P. (2011) Long-term
phenotypic correction in factor IX knockout
mice by using phiC31 integrase-mediated gene
therapy. Gene Therapy 18, 842-848. [Abstract]
- Chavez, C.L.,
Keravala, A., Chu, J.N., Farruggio, A.P.,
Gabrovsky, V.E., Voorberg, J., and Calos, M.P.
(2012). Long-term expression of human
coagulation factor VIII in a tolerant mouse
model using the phiC31 integrase system. Human
Gene Therapy 23, 390 – 398. [Abstract]
These reviews summarize some of the
recent work with phiC31 and other phage
- Karow, M. and
Calos, M.P. (2011). The therapeutic potential
of phiC31 integrase as a gene therapy system.
Expert Opin. Biol. Ther. 11, 1287-1296. [Abstract]
- Chavez, C.L.
and Calos, M.P. (2011). Therapeutic
applications of the phiC31 integrase system.
Current Gene Therapy 11, 375 - 381. [Abstract]
Another important application of
phage integrases is to create transgenic animals
and plants. We pioneered this area by showing
that phiC31 integrase could be used to target
gene addition in Drosophila embryos at high
efficiency and specificity. This method is now
popular in the Drosophila community, and related
strategies have been successful in many other
organisms, as summarized in the following
- Geisinger, J.M. and Calos, M.P. (2012).
Site-specific recombination using phiC31
integrase. In "Site-directed insertion of
transgenes.", (P. Duchateau and S. Renault,
eds.) Topics in Current Genetics Volume 23,
Springer, Chapter 8,
2013, pp 211-239. [Abstract.]
iPSC strategies for
An area of intense interest in the
lab is the use of genetically-engineered induced
pluripotent stem cells (iPSC) to develop
therapeutics. For example, iPSC derived from
patients with genetic diseases like muscular
dystrophy can be corrected, differentiated, then
engrafted back to the patient.
We have used phiC31 integrase to add
genes to reprogram adult cells into pluripotent
- Karow, M.,
Chavez, C.L., Farruggio, A.P., Geisinger,
J.M., Keravala, A., Jung, W.E., Lan, F., Wu,
J.C., Chen-Tsai, Y., and Calos, M.P. (2011)
Site-specific recombinase strategy to create
iPS cells efficiently with plasmid DNA. Stem
Cells 29, 1696 - 1704. [Abstract]
A current project involves
reprogramming fibroblasts from mdx mice, a disease model
for Duchenne muscular dystrophy, and adding the
therapeutic dystrophin gene. Corrected cells are
differentiated into muscle precursor cells in
culture and are engrafted into mice to repair
This series of six images, created
by Michele’s daughter Victoria, illustrates our
first-generation system for reprogramming and
In the first step, patient fibroblasts are
reprogrammed by co-nucleofection with a plasmid
carrying the four reprogramming genes (green
region) and a plasmid encoding phiC31 integrase.
The reprogramming plasmid also carries small
recognition sites for recombinases: an attB site
for phiC31 (blue circle, an attP site for Bxb1
integrase (blue square), and a loxP site for Cre
resolvase (purple triangle). PhiC31 integrase
(blue blob) pairs the attB site on the
reprogramming plasmid with an endogenous pseudo
attP site in the chromosomes (blue circle), to
insert the reprogramming plasmid stably into the
genome at a safe, intergenic location.
Expression of the reprogramming cassette
converts a subset of the fibroblasts into
induced pluripotent stem cells (iPSC).
The newly formed iPSC carry the reprogramming
genes and an attP recognition site for Bxb1
integrase (blue square). To restore wild-type
dystrophin to the iPSC, the cells are
co-nucleofected with a plasmid carrying the
dystrophin coding sequence, an attB recognition
site for Bxb1 integrase (square), and a loxP
site (triangle), together with a plasmid
encoding Bxb1 integrase (square blob). The
therapeutic dystrophin plasmid becomes precisely
integrated at the Bxb1 attP site in the
reprogramming plasmid resident in the
3. Excision of unwanted
The top diagram shows a close-up of the
sequences we have inserted into the chromosome.
After reprogramming is complete, the
reprogramming genes are no longer required and
are detrimental to subsequent differentiation of
the cells. Likewise, plasmid sequences that
became integrated are no longer needed or
desired. To remove the unwanted sequences, a
plasmid encoding Cre is nucleofected into the
iPSC. Cre causes precise recombination between
two loxP recognition sequences that were
engineered into the plasmid for this purpose.
After excision, only the therapeutic dystrophin
sequence, flanked by small recombinase
recognition sites, remains in the iPSC.
In vitro differentiation.
The engineered iPSC are grown in culture under
conditions that stimulate differentiation of a
significant portion of the cells into muscle
precursor cells. Such cells will bind to a
labeled monoclonal antibody that recognizes
muscle precursor cells, which include
satellite cells, the normal stem cells
resident in muscle tissue.
Fluorescence-activated cell sorting (FACS) is
used to isolate the muscle precursor cell
population from other cells in the
5. Intramuscular injection.
The sorted fraction of the differentiated
iPSC containing muscle precursor cells is
loaded into a syringe. The cells are
injected into the tibialis anterior hind
limb muscle of a mouse to assess engraftment
of the engineered and differentiated iPSC.
Injected muscles are analyzed three weeks
or more after injection to evaluate
whether engraftment has occurred.
Engraftment can be detected in several
ways. Immunocytochemistry performed on
tissue sections can be used to detect the
expression of dystrophin in the fibers.
Detection of dystrophin staining in
muscles that received engineered muscle
cells compared to uninjected muscles
suggests that engineered and corrected
iPSC have engrafted in the muscle. The
inserted dystrophin gene can also be
detected in DNA isolated from engrafted
muscle by using PCR, and dystrophin mRNA
expression can be detected by RT-PCR.