Abstract
We previously generated EGFP transgenic porcine line (Verro2GFP)
characterized by a single integration of pCAGGSEGFP
cassette, high ubiquitous EGFP expression, mendelian
transgene transmission and expression in F1 (Brunetti et al.
2008). Recently Zinc Finger Nucleases (ZFN) and TALENs
emerged as powerful tools for gene modification of different
cells types and Recombinase Mediated Cassette Exchange
(RMCE) was widely tested in different species. Moreover
EGFP-specific ZFNs were successfully used in rat (Geurtz
et al. 2010) and in pig (Watanabe et al. 2010, Whyte et al.
2010).
The purposes of our work were: a) to insert a vector suitable
for RMCE into a transcriptionally active locus and b) to assess
the possibility to obtain good quality cell colonies to be used in
Somatic Cell Nuclear Transfer (SCNT) even after two
sequential transgenic modifications (ZFN and RMCE), considering
the finite life span of Verro2GFP primary fibroblasts.
EGFP-specific ZFNs were purchased from Sigma (CompoZr®)
and the promoter-less targeting vectors were created using a
fragment of the promoter (Left-Homology-Arm = LHA) and the
polyA sequence (Right-Homology-Arm = RHA) of pCAGGSEGFP
expression vector. Cloning floxed (lox2272/lox5171)
reporter genes (PuromycinR, HygromycinR
) between these
homology sequences (LHA and RHA), we generated 2 targeting/exchanging
vectors (pB53Puro-PL, pB53Hygro-PL) and
their positive controls (C+). PCR on C+ was set up to obtain
minimal sensitivity of 100–1000 plasmid copies. Verro2GFP
fibroblasts (primary and colonies) cultured in DMEM + M199
(1:1) + FBS(10 %), 5 % CO2,5%O2, were transfected using
Nucleofector (V-24 program) and selected starting at 48 h after
transfection. Colonies were picked up at 8th selection day and
expanded in 24-well plates for PCR screening, RMCE and/or
SCNT. In ZFNs-mediated gene targeting, 2 μg of each ZFNs
coding vectors (pZFN1 and pZFN2) and 2 μg of pB53Puro-PL/
KpnI vector were used to “nucleofect” primary Verro2GFP
fibroblasts (3 105
; Puromycin = 1 μg/ml). In 3 experiments, 15
PuroR colonies were PCR screened, and 13 (87 %) were positive.
For subsequent RMCE, 4 colonies were pooled together and
4 105 cells were cotransfected with pB53Hygro-PL (2.5 μg)
and pCAG-CRE:EGFP (2.5 μg-Cre) vectors, and selected with
HygromycinB (175 μg/ml). One out of 3 HygroR colonies was positive. Finally 4 PuroR and 1 HygroR colonies were used in
zona-free SCNT to produce 243 reconstructed embryos, and 51
(21 %, 25 PuroR 26 HygroR
) blastocysts/compacted morulae
were transferred into two synchronized sows. These experiments
demonstrated that ZFN-mediated gene targeting following by
Cre-mediated cassette exchange can be successfully done during
the life span of Verro2GFP fibroblasts thus creating a feasible
platform for site-specific gene modification.
Lingua originale | English |
---|---|
pagine (da-a) | 232-233 |
Numero di pagine | 2 |
Rivista | Transgenic Research |
Volume | 22 |
Stato di pubblicazione | Pubblicato - 2013 |
Evento | Transgenic Technology
Meeting (TT2013) - , Guangzhou/Canton, P.R. China Durata: 25 feb 2013 → 27 feb 2013 |
Keywords
- transgenic swine
- zinc-finger nucleases