Generation of Hprt-disrupted rat through mouse←rat ES chimeras

We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm (green body and green sperm: GBGS rat). By injecting the GBGS rat ES cells into mouse blastocysts and transplanting them into pseudopregnant mice, rat spermatozoa were produced in mouse←rat ES chimeras. Rat spermatozoa from the chimeric testis were able to fertilize eggs by testicular sperm extraction combined with intracytoplasmic sperm injection (TESE-ICSI). In the present paper, we disrupted rat hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene in ES cells and produced a Hprt-disrupted rat line using the mouse←rat ES chimera system. The mouse←rat ES chimera system demonstrated the dual advantages of space conservation and a clear indication of germ line transmission in knockout rat production.


Production of rat spermatozoa in the mouse←rat ES chimera.
To evaluate the competency for GLT in chimeric testes, we injected six lines of rat ES cells into E3.5 mouse blastocysts and nine chimeric males were obtained from each ES cell line (Fig. 3a,b and Supplementary Table S1). None of the ES cell lines derived from F344-GBGS contributed to germ cells (Supplementary Table S1). However, two out of the three ES cell lines from Wistar/F344-GBGS contributed in testicular germ cells and rat spermatozoa were formed in chimeric testes ( Fig. 3c-e, Supplementary Table S1).
We chose one of the Wistar/F344-GBGS ES cell lines (rGBGS-ES-104) for the following experiments, as this cell line showed the highest normal karyotype rate and the most efficient GLT potency which was retained even at 16 passages (Supplementary Table S1). In the next experiment, we examined if such rat spermatozoa from the chimeric testis could fertilize eggs by TESE-ICSI 13,14 . As a result, two offspring were obtained from 93 TESE-ICSI fertilized eggs ( Fig. 3f and Supplementary Table S2).
Production of Hprt-disrupted rat using the mouse←rat ES chimera. Since the rat spermatozoa from the chimeric testes were shown to have an ability to produce normal pups, we applied this method to establish a gene-disrupted rat line ( Supplementary Fig. S2). We disrupted X-chromosome linked Hprt from ES cell lines in the conventional manner (Fig. 4a). After G418 and GANC selection, the cells were further treated with 6-thioguanine to eliminate the wild type cells with HPRT activity. We obtained three Hprt-targeted clones with normal karyotypes in more than 70% of the cells (Supplementary Table S3).
Mouse← rat ES chimeras were produced using three ES cell lines (Fig. 4b) and we found rat spermatozoa in chimeric testes from one Hprt-targeted clone (Fig. 4c, and Supplementary Table S4). The offspring were generated by TESE-ICSI from these spermatozoa (Fig. 4d) and the disruption of Hprt gene in the pups was confirmed by PCR genotyping (Fig. 4e and Supplementary Table S2). As expected, all of the female pups had the Hprt-KO allele, whereas none of the males had it.
The disappearance of HPRT protein in liver and brain, and the demise of HPRT activity (Hprt +/Y 55.9 ± 3.0, vs. Hprt −/Y , 2.7 ± 0.6 nmol/ml/h; P < 0.01) in the extract of brain homogenate were shown in Hprt −/Y rats (Fig. 5b,c). The Hprt −/Y rats were maintained for more than a year, but they showed healthy, with no signs of abnormality in their behaviour, e.g. SIB-like behaviours (Fig. 5d).

Discussion
Lesch-Nyhan syndrome (LNS) is known to be caused by a mutation of the HPRT gene leading to a deficiency or complete absence of HPRT enzyme activity. LNS patients suffer from an overproduction of uric acid which may lead to the development of uric acid crystals or stones in the kidneys, ureters, or bladder. Disorders of the nervous system and behavioural problems such as self-injury behaviour (SIB) are also common symptoms. Hprt-deficient mice have been produced to establish an animal model for this syndrome, but these animals exhibit none of the neurobehavioral abnormalities including SIB 18 . One could argue that the presence of uricase in rodents could be the cause of this difference, but the occurrence of SIB has not been affected in LNS patients when the uric acid level is lowered by xanthine oxidase inhibitors 19 .
Studies of human brains have suggested that the neurological symptoms of LNS could be related to dysfunction of the dopaminergic neurotransmitter system 20,21 . Although, the Hprt-disrupted mice showed lowered dopamine levels in the brain, SIB-like behaviour was not observed 22 . On the other hand, a forced reduction of dopamine with 6-hydroxydopamine in rats during their development is used as a model of the dopamine deficiency in LNS 23 .
Combining these facts together, we produced Hprt-disrupted rat, exploring possible production of an animal model for LNS. However, the Hprt-disrupted rats did not show any SIB-like behaviour or detrimental effect on health in the environment of our animal facilities. The role of Hprt gene in rodents (at least in mice and rats) seemed to be less essential than in human.
It is known that rat spermatogonia injected into mouse testes are supported by mouse Sertoli cells and undergo spermatogenesis to produce rat spermatozoa 24 . The fertilizing ability of these spermatozoa was proven by producing pups by microinsemination 25 . A new gene disruption method using rat germline stem (GS) cells has been explored using this xenogeneic transplantation system. Homologous recombination was successfully demonstrated in rat GS cells, but no offspring were obtained using rat spermatozoa from mouse testis even though the original rat GS cells produced fertile spermatozoa 26 . As the researchers also speculated, the karyotype of rat cells in culture, especially when exposed to a low serum concentration, is prone to develop abnormalities. In case of the rat spermatozoa derived from mouse← rat chimeric testes, it was reported that rat iPS cell-derived spermatozoa yielded offspring 27 . However, fertilizing ability of spermatozoa after homologous recombination was not examined. This is the first report that homologous recombined rat spermatozoa could be generated in the mouse← rat ES chimeric testis.
We previously reported that using a Acr3-EGFP transgene enabled observation of accumulated EGFP in sperm acrosome 17 . However, Tsukiyama et al. reported that EGFP was not detectable in rat spermatozoa derived from the mouse← rat iPSC chimeric testis using CAG/Acr3-EGFP 27 . In our experiment, we established ES cell lines from CAG/Acr3-EGFP double transgenic rats confirmed to have green fluorescent in acrosome. This enabled us to identify and retrieve rat spermatozoa despite their small number among countless mouse spermatozoa in the chimeric testis.
The phenotypes of human disease can sometimes be reproduced in KO rat more resembled manner than in KO mouse 28,29 . Therefore, if establishing a human disease animal model in mouse is difficult, it merits a try in rat. Nevertheless, reports of KO rat using ES cells through homologous recombination remain few [6][7][8][9][10] .
The reason for this has not been analysed sufficiently, but our experience and previous reports 5, 6,26 suggest that it could be attributable to low GLT ratio derived from fragile characteristics of rat ES cells in vitro cultivation. The lower the GLT ratio, the more mating pairs are required. It may also require longer breeding periods, labour, and cost. Altogether these challenge production of gene-disrupted rat. One advantage of our mouse← rat by Western blot analysis. (c) HPRT activities in the brain extract were measured by a PRECICE ® HPRT Assay Kit (#K0709-001-2). Three male rats were used in each group. Asterisk represents significant difference (P < 0.01, Student t-tests). (d) Hprt-deficient adult male rats. The upper panel shows Hprt +/Y rat, while the bottom panel shows Hprt −/Y rat. Self-inflicted scratches were not observed in Hprt −/Y and Hprt +/Y male rats.
Scientific RepoRts | 6:24215 | DOI: 10.1038/srep24215 ES chimera system is simple, early-stage identification of GLT by direct observation of rat spermatozoa in whole testis occurring as early as 10 weeks of age. Another advantage is that required animal room space almost equals that of mouse KO experiments.
Recent, genome editing technologies such as ZFNs, TALENs, and the CRISPR/Cas system function as powerful tools in production of gene modified animals 30,31 . However, for complicated gene modifications such as knock-in, conditional knockout, gene trap and chromosome engineering, ES cells may retain advantages in large-scale experiments to detect rather rare events.
Our chimeric testis method described here provides a new practical method to produce a gene-manipulated rat.

Animals.
All animal experiments were conducted in accordance with the guidelines of "Animal experiment rules" established by the Research Institute for Microbial Diseases, Osaka University, and were approved by the Animal Care and Use Committee of the Research Institute for Microbial Diseases, Osaka University. F344, and SD rats, ICR and BDF1 mice were purchased from Japan SLC, Inc. Severe combined immunodeficiency (SCID) mice (BALB/c JHan Hsd-Prkdc scid.) were purchased from Clea Japan, Inc. Wistar rats were purchased from CHARLES RIVER LABORATORIES JAPAN, INC. The double transgenic rat lines were produced by injecting a mixture of two transgenes (CAG-EGFP and Acr3-EGFP) 16,17 into the pronucleus of F344 fertilised eggs.
Construction of the targeting vector. The rat Hprt gene consists of nine exons and the targeting vector was designed to remove a part of the third exon of Hprt (Fig. 4a). A targeting vector was constructed using pNT1.1 [http://www.ncbi.nlm.nih.gov/nuccore/JN935771] 32 . For Hprt gene disruption, a 2.7 kb NotI-XhoI fragment as a short arm and a 5.7 kb KpnI-XbaI fragment as a long arm were obtained by PCR amplification using genomic DNA derived from F344 as a template. The PCR primers used were (5′ -3′ ): AAGCGGCCGCATTAGTGATGATGAACCAGGTTATGACC and TTCATGACATCTCGAGCAA GTCTTTCAGTC for the short arm (SA) and AAGGTACCTGTAGATTTTATCAGACTGAAGAGCTACTG and CTTTCCAGTTAAAGTTGAGAGATCATCTCC for the long arm (LA). DNA fragments were amplified using KOD FX (TOYOBO) for 40 cycles under the following conditions: 94 °C for 30 sec, 68 °C for 3 min (SA) or 8 min (LA). These two fragments were inserted into a pNT1.1 vector and the targeting construct was linearized with NotI.

Generation of Hprt-disrupted ES cells and genotyping. Rat ES cells were established and maintained
in 2i containing medium as described 12 . For homologous recombination, 1.6 × 10 7 rat ES cells were electroporated with 50 μg of linearized DNA in a total volume of 800 μl using a Bio-Rad Gene Pulser (250 V and 500 lF; Bio-Rad, Foster City, CA, USA). They were then plated onto 1% matrigel coated 100-mm tissue culture dishes plated with SNL cells in N2B27-2i medium. The rat ES cells were treated with 75 μg/ml of G418 at two days and 4 μM of GANC at four days after electroporation for positive/negative selection. Ten days after electroporation, 284 survival colonies were retrieved and cultured with 10 μM 6-thioguanine for obtaining of Hprt-disrupted clone (17 clones). The correct targeting was confirmed by PCR analysis for homologous recombination on both the 3′ and 5′ ends of the targeting vector.
Screening primers for ES cell clones were (5′ -3′ ): GTATGCTGGGCTCTGGACATAC and GCCTTCTAT CGCCTTCTTGACGAGTTCTTC for the short arm and CCGGTGGATGTGGAATGTGTGCGAGGCC and CTTTCCAGTTAAAGTTGAGAGATCATCTCC for the long arms. DNA fragments were amplified using Ex Taq (Takara) and Ampdirect (SHIMADZU) for 40 cycles under the following conditions: 94 °C for 30 sec, 68 °C for 3 min (SA) or 8 min (LA).
Production of the mouse←rat ES chimera. The mouse← rat ES chimeras were produced as described 12 .
Briefly, Two-or four-cell stage embryos were collected from E1.5 pregnant mice which were BDF1 females crossbred with ICR males. After incubation for 48 hours, four to six established rat ES cells were injected into each mouse blastocyst. Subsequently, mouse← rat ES chimeric blastocysts were transferred into the uterine horn of E2.5 pseudopregnant ICR mice. Mouse← rat ES chimeras were recovered by natural delivery on E19.5 or by Caesarean section on E19.5, and were identified by GFP fluorescence and coat colour.

Microinsemination.
For TESE-ICSI, male mouse← rat ES chimeric testes were obtained from animals aged ten weeks or more. After removing the tunica albuginea, seminiferous tubule fragments showing EGFP signal in the mouse← rat ES chimera were carefully dissected using fine forceps under the fluorescence microscope. GFP-positive spermatozoa extruded from the isolated tubules were suspended in Hepes-mR1ECM medium. Their unique head shapes identified rat spermatozoa. Rat spermatozoa were subsequently microinjected into oocytes as described previously 14 . On the same day, constructed eggs were transferred into the oviductal ampulla of E0.5 pseudopregnant SD rats. Offspring were recovered by natural delivery on E21.5 to E22.5 or by Caesarean section on E22.5.