Efficient gene transfer into zebra finch germline-competent stem cells using an adenoviral vector system

Abstract

Zebra finch is a representative animal model for studying the molecular basis of human disorders of vocal development and communication. Accordingly, various functional studies of zebra finch have knocked down or introduced foreign genes in vivo; however, their germline transmission efficiency is remarkably low. The primordial germ cell (PGC)-mediated method is preferred for avian transgenic studies; however, use of this method is restricted in zebra finch due to the lack of an efficient gene transfer method for the germline. To target primary germ cells that are difficult to transfect and manipulate, an adenovirus-mediated gene transfer system with high efficiency in a wide range of cell types may be useful. Here, we isolated and characterized two types of primary germline-competent stem cells, PGCs and spermatogonial stem cells (SSCs), from embryonic and adult reproductive tissues of zebra finch and demonstrated that genes were most efficiently transferred into these cells using an adenovirus-mediated system. This system was successfully used to generate gene-edited PGCs in vitro. These results are expected to improve transgenic zebra finch production.

Figure 1

Isolation and characterization of zebra finch PGCs. (A) Morphology of zebra finch PGCs cultured for a short duration in vitro. (B) RT-PCR analysis of zebra finch PGCs. Blastoderm was used as a positive control and primary ZEFs were used as a negative control. DW, distilled water. (C) Zebra finch PGCs were identified by immunostaining with an anti-DAZL antibody. (D) Scanning electron microscopy of zebra finch PGCs. (E) Migration of PGCs in recipient zebra finch embryos. Approximately 500 PGCs or ZEFs were labeled with PKH26 red fluorescent dye and injected separately into the dorsal aorta of zebra finch embryos at HH13–16. Fluorescent cells were observed in recipient embryonic gonads at HH28.

Figure 2

Enrichment and characterization of zebra finch SSCs. (A) Adult female and male zebra finches. The zebra finch indicated by the red arrow has an orange cheek patch, which is indicative of males. (B) Isolated testes of an adult male zebra finch. (C,D) Isolation of testicular germ cells by Ficoll density gradient centrifugation. Testicular cells were separated into two layers (dotted boxes). The top and bottom layers were selected and plated in culture plates. (E) Gene expression analysis of the two layers of testicular germ cells cultured for 1 day. Quantitative RT-PCR analysis of six distinct and well-accepted pluripotency marker genes (POUV and NANOG), germ cell marker genes (DDX4 and DAZL), and SSC marker genes (ITGB1 and ITGA6). Significant differences between the top and bottom layers are shown (Student’s t-test; *p < 0.05, ns = not significant). (F) Cells in the top and bottom layers were immunostained with an anti-DAZL antibody.

Figure 3

Comparison of the gene transfer efficiencies of various transfection methods in zebra finch germline-competent stem cells in vitro. (A–D) Gene transfer efficiency in zebra finch PGCs. The GFP gene was transferred into zebra finch PGCs using nucleofection, lipofection, a lentivirus, and an adenovirus. (A) Representative morphology of transfected zebra finch PGCs. (B,C) Transfection efficiencies measured by flow cytometry. Significant differences between the four groups are shown (one-way ANOVA; **p < 0.05; ns = not significant). (D) The percentage of surviving cells was determined by trypan blue staining. Significant differences between the four groups are shown (one-way ANOVA; *p < 0.05; **p < 0.005; ns = not significant). (E–H) Gene transfer efficiency in zebra finch SSCs. (E) Representative morphology of transfected zebra finch SSCs. (F,G) Transfection efficiencies measured by flow cytometry. Significant differences between the four groups are shown (one-way ANOVA; ***p < 0.0001; ns = not significant). (H) The percentage of surviving cells was determined by trypan blue staining. Significant differences between the four groups are shown (one-way ANOVA; ***p < 0.0001; ns = not significant).

Figure 4

CRISPR/Cas9-mediated gene targeting using the adenovirus-mediated method in zebra finch PGCs. (A) Schematic of the gRNA targeting the zebra finch ADNP gene. The gRNA sequence located in exon 6 is shown in red letters and the PAM sequence is shown in light blue letters. (B) Construction of the CRISPR/Cas9 adenoviral vector containing the selected gRNA sequence targeting the ADNP gene. (C) CRISPR/Cas9 adenovirus-transduced zebra finch PGCs. GFP expression indicates transduced cells. (D,E) Knockout efficiency in zebra finch PGCs according to the T7E1 assay and DNA sequencing. Total PCR products were inserted into the T-vector and analyzed by DNA sequencing. Red letters indicate gRNA recognition sequences, light blue letters indicate PAM sequences, and gray letters indicate deletions. The dotted box indicates the Tyr719* residue located in exon 6 of the ADNP gene, a major point mutation causing ADNP syndrome. (F,G) In vivo migration of genome-modified zebra finch PGCs in recipient embryos. Approximately 1000 cells transduced with the adenovirus carrying the CRISPR/Cas9 system were labeled with PKH26 red fluorescent dye, injected into the dorsal aorta of zebra finch embryos at HH13–16, and incubated until HH28. Fluorescent cells were observed in recipient gonads and counted. (H) Some recipient gonads were paraffin-sectioned and immunostained with an anti-GFP antibody.