Abstract
BackgroundSmall animals that show a deficiency in klotho exhibit extremely shortened life span with multiple aging-like phenotypes. However, limited information is available on the function of klotho in large animals such as pigs.ResultsIn an attempt to produce klotho knockout pigs, an sgRNA specific for klotho (targeting exon 3) was designed and Cas9-sgRNA ribonucleoproteins were transfected into porcine fibroblasts. Transfected fibroblasts were cultured for one to 2 days and then directly used for nuclear transfer without selection. The cloned embryos were cultured in vitro for 7 days and analyzed to detect modifications of the klotho gene by both T7E1 and deep sequencing analysis. Modification succeeded in 13 of 20 blastocysts (65%), 8 of which (40.0%) were monoallelic modifications and 5 (25.0%) were biallelic modifications. Based on high mutation rates in blastocysts, we transferred the cloned embryos to 5 recipient pigs; 1 recipient was pregnant and 16 fetuses were recovered at Day 28 post transfer. Of the 16 fetuses, 9 were resorbing and 7 were viable. Four of 9 (44.4%) resorbing fetuses and 3 of the 7 (42.9%) viable fetuses had monoallelic modifications. Thus, 3 klotho monoallelic knockout cell lines were established by primary culture. A total of 2088 cloned embryos reconstructed with 2 frame-shifted cell lines were transferred to 11 synchronized recipients. Of the recipients, 7 of 11 eleven (63.6%) became pregnant. However, none of the pregnancies was maintained to term. To discover why klotho monoallelic knockout fetuses were aborted, expression of aging- and apoptosis-related genes and klotho protein in placentas from klotho monoallelic knockout and wild-type fetuses was investigated. Placentas from klotho monoallelic knockout fetuses showed negatively changed expression of aging- and apoptosis-related genes with lower relative expression of klotho protein. These results indicated that the reason why klotho monoallelic knockout fetuses were not maintained to term was possibly due to decreased klotho expression in placentas, negatively affecting aging- and apoptosis-related genes.ConclusionsKlotho monoallelic knockout porcine fetal fibroblasts were successfully established. However, pigs carrying klotho monoallelic knockout fetuses failed to maintain full-term pregnancy and a decrease in klotho expression in placenta likely leads to pregnancy loss.
Highlights
Small animals that show a deficiency in klotho exhibit extremely shortened life span with multiple aging-like phenotypes
clustered regularly interspaced short palindromic repeat (CRISPR)-mediated genome editing in pigs is generally accomplished by somatic cell nuclear transfer (SCNT) with donor cells transfected with single-guide RNA and Cas9 DNA [4]
This study demonstrated klotho gene modifications in cloned fetuses generated by transfer of embryos reconstructed with non-selected Cas9-single-guide RNA (sgRNA) transfected fetal fibroblasts
Summary
Small animals that show a deficiency in klotho exhibit extremely shortened life span with multiple aging-like phenotypes. Recent advances in genome editing techniques such as Zinc-Finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas9) system have enabled the production of animal models for specific purposes [2]. Transfection with plasmid DNA encoding sgRNA and Cas is limited by off-target effects and unwanted integration of DNA segments at both on-target and off-target sites in the genome [5]. To overcome this issue, delivery of Cas9-sgRNA ribonuleoproteins (RNPs) into cells or embryos has been used. Delivery of pre-assembled Cas9-sgRNA RNPs facilitates highly efficient genome editing in cells, embryos, and organisms [6,7,8]
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