Transgenic quail production by microinjection of lentiviral vector into the early embryo blood vessels

Abstract

Several strategies have been used to generate transgenic birds. The most successful method so far has been the injection of lentiviral vectors into the subgerminal cavity of a newly laid egg. We report here a new, easy and effective way to produce transgenic quails through direct injection of a lentiviral vector, containing an enhanced-green fluorescent protein (eGFP) transgene, into the blood vessels of quail embryos at Hamburger-Hamilton stage 13-15 (HH13-15). A total of 80 embryos were injected and 48 G0 chimeras (60%) were hatched. Most injected embryo organs and tissues of hatched quails were positive for eGFP. In five out of 21 mature G0 male quails, the semen was eGFP-positive, as detected by polymerase chain reaction (PCR), indicating transgenic germ line chimeras. Testcross and genetic analyses revealed that the G0 quail produced transgenic G1 offspring; of 46 G1 hatchlings, 6 were transgenic (6/46, 13.0%). We also compared this new method with the conventional transgenesis using stage X subgerminal cavity injection. Total 240 quail embryos were injected by subgerminal cavity injection, of which 34 (14.1%) were hatched, significantly lower than the new method. From these hatched quails semen samples were collected from 19 sexually matured males and tested for the transgene by PCR. The transgene was present in three G0 male quails and only 4/236 G1 offspring (1.7%) were transgenic. In conclusion, we developed a novel bird transgenic method by injection of lentiviral vector into embryonic blood vessel at HH 13-15 stage, which result in significant higher transgenic efficiency than the conventional subgerminal cavity injection.

Figure 1. Diagram of the relevant regions of the pGCL-eGFP vector used to generate chimeric quail.

LTR, long terminal repeat; Psi, packaging signal; RRE, rev responsive element;U6,U6 promoter; CMV, cytomegalovirus promoter; eGFP, enhanced green fluorescent protein gene; WRE, woodchuck hepatitis virus post-transcriptional regulatory element; SinLTR, self-inactivating LTR. The Southern blot probe and PCR product to detect the eGFP gene are indicated. PstI and EcoRI restriction enzymes were used for Southern blot analysis.

Figure 2. Expression of eGFP in tissues of hatched transgenic chimeric quail by fluorescence microscopy.

Lentiviral vector was injected into the blood vessels of HH13–15 embryos. eGFP expression in (A) the vitelline membrane of 4 day-old embryo; and in (B) the beak, (C) feather, (D) liver, (E) kidney, (F) mesenterium, (G) glandular stomach, (H) small intestine, (I) large intestine, (J) blood vessel, (K) breast muscle, (L) spleen, (M) heart, (N) oviduct, (O) eye, and (P) brain of newly hatched G0 quails; and in (Q) the follicle of sexually matured G0 quail, (R) the skeletal muscle of G1 progeny.

Figure 3. Expression of eGFP in the gonads.

Lentiviral vector was injected into the blood vessels of HH13–15 embryos. Newly hatched quails were observed by fluorescence microscopy. (A, B, C, D) Gonads of a 3 day-old male quail left and right. (E, F) Enlargement of part of the gonads. (B, D, F) are bright-field images.

Figure 4. PCR analysis of genomic DNA extracted from semen of transgenic germ line chimeric (G0) quail.

(A) PCR product of eGFP; (B) PCR product of housekeeping gene GAPDH; (1–5) PCR products of sexually matured G0 males produced by microinjection to blood vessels of embryo at HH Stage 13–15 (6–8). PCR analysis of three sexually mature males produced by microinjection beneath the subgerminal cavity of the blastodermal embryo. (M) Standard DNA markers. (P) Positive control. (N) negative control without microinjection of lentiviral vector. PCR product size is indicated on the right.

Figure 5. Expression of eGFP in the ovary.

Lentiviral vector was injected beneath the subgerminal cavity of blastodermal embryos. Newly hatched quails were observed by fluorescence microscopy. (A, C) Ovary of a 3 day-old female quail. (B, D) Enlargement of part of the ovary. (C, D) are non-transgenic control.

Figure 6. PCR and Southern blot analysis of genomic DNA from the blood of G1 transgenic quails.

(A) PCR analysis of G1 offspring obtained by microinjection to the subgerminal cavity of blastodermal embryos. Lanes 1–4 represent the transgenic quails. (B) PCR analysis of five G1 offspring produced by microinjection into the blood vessels of HH13–15 embryos. Lanes 1–6 represent transgenic quails 1–6; MW, DNA size markers; P, positive control; N, negative control; PCR product size is indicated on the right. (C) Southern blot results. N, nontransgenic quail; Lanes 1–4 indicate G1 transgenic quails 1–4 (as in A). (D) Genomic DNA (10 µg) was digested with PstI and EcoRI and hybridized with the eGFP probe. P, 80 pg of pGCL-eGFP vector; N, negative control without injection of lentiviral vector; Lanes 1–6 indicate G1 transgenic quails 1–6 (as in B).

Figure 7. Fluorescent images of the lungs from quails.

(A) the lung from a G0 quail from blood vessel injection group; (B) the lung of a non-injection control.