The study procedures were confirmed by the Research Ethics Committee of Shahid Beheshti University medical of Sciences, Tehran, Iran (IR.SBMU.MSP.REC.1395.5.1) Also all experiments were performed in accordance with relevant guidelines and regulations.
Gene-targeted ES cells
Biallelic RAG-1 knock-out mES clones (strain 129) had been produced using CRISPR/Cas9 gene editing system and cultured according to the protocols described in our previous work (13). Briefly, mESCs were grown on 0.1%-gelatin-coated culture flask in the absence of feeder cells in mESC culture medium supplemented with R2i and 2% ES-FBS. Transfection of mESCs was done using Lipofectamine 2000 according to manufacturer’s instructions. Single clones of mESCs were obtained by serial dilution in 96-well plates. After clone screening and validation, the pluripotent state of the mutant ES cells was confirmed by Quantitative analysis of pluripotency markers, Nanog, Oct4, and Sox2 genes (13).
This study was carried out in compliance with the ARRIVE guidelines. 6 to 8 weeks old C57BL6 (embryo donors), CD1 (recipients) and C57BL6 (breeders) mice were purchased from pasteur institute of Iran (Karaj, Iran).All strains were housed in standard conditions with a 12 h on/12 h off light cycle, 18 to 20°C temperature and standard mouse diet with autoclaved water. The care and use of all mice were approved by the animal care and use protocol of pasteur institute of Iran.
Media and chemicals
Pregnant mare serum gonadotropin (PMSG) folligon was purchased from MSD Animal Health (www.msd-animal-health.co.in). Human chorionic gonadotropin (HCG/pregnyl) was directly purchased from pharmacies of Tehran (Tehran, Iran). M2 and KSOM embryo culture media, Polyvinylpyrrolidone (PVP360), Bovine serum albumin (BSA, embryo tested), Embryo culture-tested mineral oil, 1,4-Dithiothreitol (DTT) and L-Glutamine were purchased from Sigma-Aldrich (www.sigmaaldrich.com). Trehalose dehydrate and skim milk were purchased from Merck (www.merckmillipore.com). Ketamine 10% /Xylazine 2% (Alfasan, Netherland) anesthetics mix was freshly prepared.
SYBR Premix Ex TaqII reagent (Takara Bio, Kusatsu, Shiga, Japan) was purchased from Takara (www.takarabio.com).
Embryo collection and mESCs injection
Six days prior to ES injection of embryos, superovulation of C57BL6 mice was induced by IP injection of 7.5 IU PMSG and 48 h later, by IP injection of 7.5 IU hCG. Blastocysts were collected from uterus on 3.5 pdc in M2 medium from natural mating of superovulated mice and Morulae were isolated from oviducts on 2.5 pdc. Collected embryos were cultured in KSOM medium with amino acids (KSOMaa) at 37 °C in 5 % CO2 before mESCs injection.
Two days prior to ES injection, Rag1 knocked-out ES cells which had been stored in liquid nitrogen, were thawed and cultured in gelatin-coated flasks at 37 °C in5 % CO2.
ES injection of host blastocysts or morulae from C57BL6 strain was performed in M2 medium with 12-15 ES cells into blastocysts and 8-9 ES cells into morula using laser and micromanipulator.
In the current study, the blastocysts isolated from natural mating were considered as the sham group. The control group included the blastocysts isolated from in vivo-derived morula, while the Rag1 knocked-out blastocysts developed by microinjection of Rag1 knocked-out mESCs into the morula or blastocyst were defined as the test group.
The injected embryos in comparison with the non-manipulated embryos were evaluated for the expression of pluripotency, trophectoderm, and imprinting genes using real-time PCR and for the methylation rates of H3K9me3 and H3K4me3 using Immunohistochemical analysis.
A number of injected (Rag1 knocked-out) blastocysts were transferred into the uterus of recipients for producing chimeric mice and testing GLT.
Real time PCR
RNA extraction and cDNA synthesis
The real time PCR was performed to evaluate the expression levels of TEAD4, Oct-4, Cdx2, Nanog, H19, and Igf2 genes (Table.1). Total RNA was isolated from 5 blastocysts by the RNA extraction kite (Life Technologies, Gent, Belgium) based on the manufacturer's instructions. The RNA concentration of each sample was measured by a spectrophotometer (Pico drop Real-Life), and then, the extracted RNAs were suspended in 10 μl of DEPC water and stored at -80 ℃, until they were used for cDNA synthesis. In the next step, cDNAs were synthesized based on the random hexamer approach using Prime Script Quanti Tect Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. The following thermocycling condition was considered to synthesis cDNAs: 2 min at 42 ºC, 15 min at 42 ºC followed by 3 min at 95 ºC. Finally, the synthesized cDNA was stored at 20 °C.
The blastocysts in each experimental group were fixed in 4% paraformaldehyde for 1 h at 4-8 ℃. Then, the blastocysts were placed at PBS containing 0.1 % Tween 20 for 30 min and kept in 0.1% Triton at 4-8 ℃. After washing, the blastocysts were placed in PBS containing 2% BSA and kept in incubator for 1 h to black antigens of blastocysts. At the next step, the blastocysts were treated with the primary antibodies for 1 h at the room temperature, followed by 1 h incubation with the secondary Alexa Flour 594 antibodies at the incubator. Finally, the cell nucleuses were stained using DAPI.
Gene expression evaluation
Following cDNA synthesis, the real time PCR was carried out on cDNA samples using SYBR Premix Ex TaqII reagent (Takara Bio, Kusatsu, Shiga, Japan) to measure the expression rate of the genes involved in embryonic development. Thermal cycling was carried out on a Rotor-Gene Q instrument (Qiagen, Hilden, Germany) under the following conditions: 30 s at 95°C, 50 cycle of 5 s at 95°C, 30 s at 60°C, 60 to 95 °C with a ramp rate of 0.3°C/s as melting curve. To normalize the relative expression rates of the aforementioned genes, the expression of H2afz and GAPDH were measured as the housekeeping genes. The REST software was used to analyze the results and p ≤0.05 was considered to be meaningful.
Chimera production, breeding and GLT testing
After injecting embryos, they were transferred into the uterus of 2.5 dpc CD1 recipients in order to produce chimeric mice. Chimeras were examined at 12 days of age for coat chimerism.
For testing GLT, male chimera at 8 weeks of age were bred with wildtype female C57BL/6N mice (male chimera are set with two females per cage). This process was repeated several times for each male chimera. In the case of successful pregnancy and delivery, pups were assayed for the agouti dominant coat color and RAG1 mutation analysis by PCR and Sanger sequencing for the presence of mutations. If the agouti coat color was not observed in 40 pups per male chimera, it indicates that the RAG1 knockout ES cell did not participate in the germ cell layer, so GLT was not successful.
In the absence of pregnancy after 4 weeks, females were replaced with new females. If pregnancy did not occur after 50 days, male chimera infertility in comparison with a fertile male was assessed by sperm collection and analysis of quality and genotype of collected sperms.
Sperm collection, DNA extraction and PCR reaction
Male mice were anesthetized with appropriate dose of ketamine and xylazine. After incision at the site around the base of the penis, the fat pad with the testicles, epididymis and vas deferens, were pulled out. Then, using a transfer needle (internal diameter of 120 μm), connected to the injection pump, the sperm sample was removed after inserting a small amount of PBS buffer into the vas deferens. If the vas deferens tubes were empty of sperm, the sample was expelled from cauda epididymis. Sperm samples were emptied into a tube containing PBS buffer. Sperm morphology and motility were assessed under the phase contrast microscope.
Sperm DNA extraction was performed according to a simple improved kit-based protocol in which DNA extraction was efficient even from a small amount of sperm (~20,000 sperms) (Figure 5, Figure S5).
In this method, sperm samples diluted in PBS to a final volume of 200 µL. Sperm suspensions were pulse-vortexed for 5 min in 200 µL lysis buffer (Favorgen Biotech, taiwan) with additives including reducing agent DTT (final concentration: 150 mM), 0.5% Triton X-100 and Proteinase K (final concentration: 200 µg/mL), then incubated at 56°C for 4 h and vortexed vigorously every 1 h. In the following, DNA extraction was performed according to the protocol of DNA isolation kit (Favorgen Biotech) and PCR reaction was performed with RAG-1 primers (forward primer: GAA GAA GCA CAG AAG GAG AAG, reverse primer: ATC GGC AAG AGG GAC AAT AGC) under the same condition described in previous protocol (13).
Sperm cryopreservation in Cryoprotective medium (12% trehalose dehydrate, 3% skim milk, 100 mM L-Glutamine) was performed in liquid nitrogen according to the protocol described by Takeo et al. (14). Briefly, 100 μl of Cryoprotective medium was poured onto the epididymis, several incisions were made in the cauda epididymis and the plate was shacked and incubated at 37 °C for 10 min in order to release sperms into the medium. The entire medium containing the sperm was then collected and transferred to the freezing straws. The sperm straws were placed above liquid nitrogen for 10 minutes and quickly plunged in the liquid nitrogen. After a few days, in order to assess the viability and motility of the frozen sperms, sperm straws were removed from the liquid nitrogen and immersed in 37 ° C water for 10 minutes. Almost right after thawing cryopreserved C57BL/6 mouse sperms as control, 30% of sperms were highly mobile and the viability was 40%.