Efficient KO of ovine B-lactoglobulin (BLG) gene and KI of recombinant human factor IX (rhFIX) under BLG native regulatory sequences both in somatic cells and zygotes using TALEN nucleases

BEVACQUA RJ; CARLSON DF; FERNANDEZ-MARTÍN R; GIBBONS A; SAVY V; CANEL NG; VANS LANDSCHOOT G; DE LA ROSA L; LANGE F; ALBERIO V; BRISKI O; GISMONDI MI; TABOGA O; FERRARIS S; FAHRENKRUG SC; SALAMONE DF

Bangkok

Congreso; 44th IETS Annual Conference; 2018

IETS

Site-specific genetic engineering is a valuable tool for pharmaceutical research and development of biomedical models. Despite engineered nucleases allow targeted gene edition in a rather simple fashion; few reports are available so far on specific knock in (KI) combined with engineered nucleases in domestic species. Here, we evaluated the possibility of inducing specific KI of cDNAs coding for proteins of pharmaceutical interest under the control of milk native promoter sequences, taking advantage of the TALEN system. We designed two TALEN pairs, targeting exon 1 and 5 of ovine BLG, and a homologous recombination vector, harboring rhFIX cDNA, to be introduced under BLG regulatory sequences. In an initial set of experiments, 5x105?1x106 ovine fibroblasts were transfected with 1 μg of each TALEN mRNA. When corresponding, the plasmid for homologous recombination (HR) (pHR-hFIX) was also included at 50 ng/μl. The feasibility of inducing KO was confirmed by Cel1 assay. The deletion of the entire genomic region among TALEN target sites and the occurrence of HR in cell lysates were detected by PCR. Also, 576 individual colonies were picked up and PCR analyzed. The deletion of the region between TALEN target sites was achieved with 0.1% efficiency. The incidence of HR in cells was 0.005%, as detected by PCR. For zygotes, laparoscopic artificial insemination (AI) was performed on synchronized and superovulated ewes. Zygotes were recovered 16 h after AI. Three different cytoplasmic injection conditions were compared: i) 5 ng/μl TALEN mix (2.5 ng/μl oaBLG T1.1 + 2.5 ng/ μl oaBLG T5.1) (5TM); ii) 5 ng/μl TALEN mix combined with 25 ng/μl pHR-hFIX plasmid (5TM+25pRH) and iii) 15 ng/μl TALEN mix (7.5 ng/μl oaBLG T1.1 + 7.5 ng/ μl oaBLG T5.1) combined with 50 ng/μl pHR-hFIX (15TM+ 50pRH). A non-injected control (NIC) was also included. Embryo analysis was conducted on whole-genome-amplified DNA from blastocysts, followed by PCR and sequencing. Although blastocyst rates for NIC and 5TM did not statistically differ, 5TM+25pRH and 15TM+50pRH groups resulted in lower blastocysts rates than the NIC [p