Experimental design
Experiment 1 was to optimize the protocol for generation of transgenic mice by lentiviral transduction of male germline stem cells. The effects of injection sites and age of pre-founder mouse was to be elucidated. Experiment 2 was to detect the effect of the promoter for driving the expression of the transgene.
Animals
C57BL/6J male mice (aged 7 d and 28 d) and C57BL/6J female mice (aged 1.5 to 2 mo.) were obtained from the Fourth Military Medicine University (Xi’an, China). All mice were supplied with water and chow ad libitum, housed and bred in a sterile environment with the controlled temperature (25 ± 5 °C) and humidity (30–70 %), and 14 h night a day. The day of birth was designated as d 0. All protocols for the experiments were approved by and performed under the guidance of the Institutional Animal Care and Use Committee of the Northwest A&F University. All the treatments were carried out in accordance with relevant guidelines and regulations.
Viral vectors
Lentiviral vectors as a type of retrovirus that can infect both dividing and non-dividing cells were widely used for introduce transgenes or gene knockdown. The non-dividing or terminally differentiated cells such as neurons, macrophages, hematopoietic stem cells, retinal photoreceptors, and muscle and liver cells can be transduced by lentiviral vectors [17]. The lentiviral vectors containing cytomegalovirus (CMV) or human elongation factor-1 (EF-1) promoters to drive eGFP expression were used in this study. The CMV promoter is a widely used promoter and EF-1 is a constitutive promoter of human origin. The lentivirus with EF-1 promoter driving eGFP was packaged in our lab based on the previous protocols [18], and the lentivirus with CMV promoter driving eGFP was a gift from Dr. Wuzi Dong (College of Animal Science and Technology, Northwest A & F University).
Injection of lentiviral particles and generation of transgenic mice
In Experiment 1, to test age effect of pre-founder, sixteen male mice aged 7 d (body weight 4 ± 1 g) were randomly allocated to two groups, named A and B. And sixteen males aged 28 d (body weight 14 ± 3 g) were randomly allocated to another two groups, named C and D group.
The 7-day-old mice were anaesthetized by intraperitoneal injections of Avertin (400μL/30 g body weight). An incision of approximate 0.3–0.5 cm in length was made in skin and muscle anterior to the penis with a sterile ophthalmology scissors. With the help of a sterile dressing forceps, the testes were moved out from the enterocoelia or scrotal sac gently and the dorsal fat pad was pulled for easy operation. To elucidate an effect of injection site, the lentivirus particles were re-suspended in PBS buffer (Life Technology) with trypan blue (0.04 %). For Group A/C, lentivirus was injected directly into inter-seminiferous tubular space using a syringe with a needle. For Group B/D, lentivirus was injected into seminiferous tubules via efferent ducts or rete testis under a stereoscopic microscopy as described by Shirohana [19]. The pre-founders were implemented artificial feeding for at least 1 wk, then were fed by a maternal mouse for 2 wk followed by self-help feeding. At 6 wk after injection, these pre-founder mice were mated with wild type females of the same strain (aged 1.5–2 mo).
The 28-day-old mice were injected with a higher dose of Avertin (500 μL/30 g body weight) by intraperitoneal, the inguinal area hairs were removed and iodine was used for clean and disinfectant. In an effort to avoid injury to the penis, a wound from skin to muscle anterior to the penis of approximate 0.5–1.0 cm length was cut on a sterile bench. Lentivirus was injected as described above.
Five microlitters of the lentiviral particles at the concentration of 5.5 × 106 TU/mL were used in each injected injection in of this study for 7-day-old mice, and ten microlitters of the lentiviral particles for older ones. All the pre-funders were named 0001, 0002 and so on. In Experiment 2, based on the results of Experiment 1, 10 μL of lentiviral vectors (5.5 × 106 TU/mL) were injected into inter-tubular space of a C57BL/6 J male mice (aged 28 d, n = 3). At 5 wk after injection, these pre-founder mice were mated with wild type females of the same strain (aged 1.5–2 mo).
Genotyping
At 5 or 6 wk post-surgery, the pre-founder male mice were mated with mature wild-type female mice. At 4 wk post-coitum, the new pups of F1 generation were genotyped by PCR in Experiment 1. The eGFP specific primers (Table 2) were used for genotyping. Only those pups with eGFP specific band were considered as transgenic F1 and were named as 1001, 1002 and so on. In order to test whether the transgene could pass to the next generation, half of F1 generation was mated with each other, and the other half was mated with wild-type for F2 generation. The F2 generation pups were named 2001, 2002 and so on.
Isolation of Genomic DNA (gDNA)
The tail tips (approximately 0.5 cm in length) from F1 and F2 pups and testis tissue from pre-founders were collected for DNA extraction. Samples were cut into small pieces and lysed for 16 h at 55 °C in a high salt digestion buffer containing 50 mmol/L Tris · HCl, 1 % SDS, 100 mmol/L NaCl, 100 mmol/L EDTA and 1,200 μg/ mL Proteinase K (Tiangeng, China). The lysate was processed for extraction of DNA based on salting-out methods as described [19].
Polymerase Chain Reactions (PCR) and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Ubiquitous gapdh gene was amplified as a loading control. The pCD513B-CMV-MCS-EF1 plasmid DNA was introduced as a positive control while the genomic DNA obtained from wild-type mice as a negative control. RNA was isolated from transgenic F1 and F2 animals with TRIZOL (Life Technology, USA) and reverse transcribed (RT) to cDNA with Superscript III RT First Strand cDNA synthesis Kit (Invitrogen, USA) according to the manufacturer’s protocol. PCR was performed in duplicate in a 20 μL of reaction volume consisting of TaKaRa Ex Taq (TAKARA, Japan), 0.5 μmol/L of each primer and 500 ng of gDNA or cDNA. The PCR protocol included one cycle at 94 °C for 10 min, 32 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s, then followed final extension for 10 min at 72 °C. The PCR products were visualized after 2 %-agarose gel electrophoresis stained with ethidium bromide (Sigma-Aldrich, USA).
Western blot
Proteins of the interest tissues (heart, skin, liver, muscle and testis) were isolated from transgenic mice that were confirmed by RT-PCR (non-transgenic pups as negative control). The protein concentration was determined using a Quick Start™ Bradford Protein Assay Kit (Bio-Rad, USA). Protein complexes were separated by SDS-PAGE, and transferred to nitrocellulose membranes (Hybond ECL, USA). Membranes were probed using the following primary antibodies: anti-β-actin (Abcom, 1: 1,000), anti-GFP (Abcom, 1: 1,000). Secondary antibodies were horseradish peroxidase-linked anti-rabbit antibody (Santa Cruz, 1: 2,000). Protein bands were visualized on a Bio-Rad Chemidoc XRS using a Western Bright ECL Kit (Advansta, Menlo Park, CA, USA).
Histology
The testis tissue from transgenic mice (aged 2 mo) in Experiment 2 was collected and fixed in Bouin’s solution overnight, embedded in paraffin for serial sectioning at 5–7 μm. The sections were stained with hematoxylin and eosin (H.E., Sigma-Aldrich, USA) and viewed under the light microscope (Olympus, Japan).
Bisulfite sequencing PCR
The genomic DNA from the testis tissue of transgenic F1 mice (pre-founder transduced with lentivirus with promoter CMV or EF-1) was directly subjected to bisulfite conversion with EZ DNA Methylation Direct kit (Zymo Research, Orange, CA, USA) according to the manufacturer's protocol. The BSP specific primers (Table 2) were used for bisulfite-modified DNA amplification. After bisulfite conversion, the PCR products were cloned into a pGEM-T Easy Vector (NEB, England), and nine individual clones with different promoters were sequenced by BIO5 Institute, University of Arizona.