Utilization of central disk of blastoderm and germinal crescent region for production of interspecific germline chimera between chicken and quail

Tomoki Soh¹, Yoshie Inoue¹, Yong-Mei Xi¹, Yukio Kato², Masa-Aki Hattori¹

Asian J Androl  2002 Jun; 4:  83-86             

Keywords: chicken; quail; chimera; germline; semen; polymerase chain reaction

Abstract

Aim: The production of interspecific germline chimeras between chicken and quail were attempted employing the dissociated cells derived from the blastodermal central disk (stage X) and the germinal crescent region of embryo (stage 7-8). Methods: The central disk (CD) of the area pellucida in chicken blastoderm (stage X) and the germinal crescent region (GCR) of embryo (stage 7-8) were dispersed and injected into the subgerminal cavity of quail blastoderm (stage X). Injected eggs were incubated for 7 days or to hatching. The donor chicken DNA was detected by the polymerase chain reaction. Results: In day-7 embryos, chicken DNA was detected in 5 gonads and 9 brains from 53 survived embryos received chicken CD cells, and 1 gonads and 6 brains from 27 survived embryos received chicken GCR. Chicken DNA was also detected from the semen of one adult male hatched from eggs received chicken GCR cells. Conclusion: CD and GCR cells as the donors showed the possibility to produce the interspecific germline chimera, but further studies are needed to make necessary improvement.

1 Introduction

With the purpose of the preservation of endangered species and conservation of precious fundamental species in bird, the production of interspecific germline chimeras has been produced [1-5]. However, up to date its efficiency is low and the progeny test entails a lot of work. In order to facilitate the improvement of this method, the gene marker that can detect the donor cells in recipient is employed as a very important tool. There are several gene markers for interspecific chimeras between chicken and quail [6,7]. In the present study we have made use of another primer set for chicken ovalbumin to identify the donor chicken DNA in the recipient quail by polymerase chain reaction (PCR).

Primordial germ cells (PGCs) are the most available cells to produce germline chimera [2-4,6,8-14]. PGCs originate in the central disk (CD) of blastoderm at stage X [15-23] and move to the germinal crescent region (GCR) at stage 7-8 [16,24-26]. In the present study, we used the cells of CD in chicken blastoderm at stage X [15] and GCR of chicken embryo at stage 7-8 [24] to produce chicken-quail interspecific germline chimera because the CD and GCR cells were easily obtained.

2 Materials and methods

2.1 Donor cells

Fertilized chicken eggs, freshly oviposited (stage X [15]) and incubated for 26-29 h (stage 7-8 [24]), were swabbed with 70 % ethanol. CD cells of the area pellucida in the blastoderm (stage X [15]) and GCR (stage 7-8 [24]) were cut and dispersed in phosphate buffered saline (PBS). The cells were resuspended in Dulbecco's MEM to make a final concentration of 500 cells/mL through removing the supernatant after centrifugation (400g, 10 min).

2.2 Recipient eggs and injection

Fresh and fertile quail eggs (stage X [15]) swabbed with 70 % ethanol were stored for 2~3 days at room temperature with the marked equatorial plane upward. A window 5 mm in diameter was opened at the mark. Then 1 mL of donor cell suspension (500 cells) was injected into the subgerminal cavity of the recipient embryo using drawn micropipette (50-70 mm outer diameter). The window was closed with a piece of adhesive tape. Injected eggs were incubated for 7 days or to hatching at 37.7 and 70 % relative humidity.

2.3 Preparation of genomic DNA

The gonads and a part of the brain were collected from the embryos of day-7. Semen was collected from adult male quails hatched from eggs received GCR cells. Genomic DNA samples were extracted by the conventional method using phenol-chloroform. The DNA concentration was determined by spectrophotometer at 260 nm.

2.4 PCR

To detect the chicken genomic DNA by PCR, a primer set for ovalbumin cDNA (GenBank #V00383) was designed: primer C1, 5'-GAGTGGATGCTGCAAGCGT-3' (sense) and primer C2, 5'-CTGTGAGTCCAA-TTTCATTCCCT-3' (antisense). After an initial denaturation step (95 , 10 min), the PCR was performed in a reaction mixture (10 mL) containing 0.2 mM each of the primers, 1 PCR buffer, 0.2 mM dNTPs, 0.25 U AmpliTaq Gold polymerase (Perkin Elmer Applied Biosystems, Foster,USA), and 10 ng of DNA template. The amplification step consisted of 40 cycles denautration (95 , 30 sec), annealing (60 , 30 sec) and extension reaction (72 , 2 min) before a final extension step of 10 min at 72 . The PCR products, with a predicted size of 357 bp (Figure 1), were analyzed by electrophoresis on 1.5 % agarose gels. The PCR products amplified with quail genomic DNA was checked by a restriction enzyme BsiHKA I (New England BioLabs, Beverly, USA), and the resulting products were analyzed by electrophoresis on 1.5 % agarose gels. To compare the intensity of the signals, 10, 1 and 0.1 ng of chicken genomic DNA with 10 ng of quail DNA were used as the template. The quail genomic DNA was used as the negative control.

Figure 1. Nucleotide sequence of the amplified region of chicken ovalbumin cDNA (GenBank #V00383). Underlined sequences are the primer regions.

1081             gagtggatgc  tgcaagcgtc   tctgaagaat

1141 ttagggctga   ccatccattc   ctcttctgta    tcaagcacat  cgcaaccaac  gccgttctct

1201 tctttggcag    atgtgtttcc    ccttaaaaag  aagaaagctg aaaaactctg   tcccttccaa

1261 caagacccag agcactgtag  tatcaggggt   aaaatgaaaa gtatgttctc    tgctgcatcc

1321 agacttcata   aaagctggag  cttaatctag   aaaaaaaatc agaaagaaat  tacactgtga

1381 gaacaggtgc  aattcacttt     tcctttacac   agagtaatac  tggtaactca  tggatgaagg

1441 cttaagggaa   tgaaattgga   ctcacag     

3 Results

3.1 Verification of the PCR product

The PCR product of the expected size (357 bp) was amplified from chicken genomic DNA, and when the template was decreased, the intensity of the signal was weakened (Figure 2). The quail genomic DNA was not amplified with the primer set for chicken. The 357 bp chicken PCR fragment was cleaved with BsiHKA I, which yielded the expected fragments of 193 and 164 bp.

3.2 Detection of chicken DNA in day-7 quail embryo

A total of 215 quail eggs received chicken CD cells. The survival rate of embryos at day-7 was 25 % (Table 1). Chicken DNA was detected from 5 embryos in the gonad and 9 embryos in the brain as revealed by PCR analysis. A total of 184 quail eggs received chicken GCR cells. The survival rate of embryos at day-7 was 15 %. Chicken DNA was detected from 1 embryo in the gonad and 6 embryos in the brain. The intensity of the PCR signals of embryos received CD or GCR cells was estimated around 1/100 comparing with the positive control.

Table 1. Detection of chicken DNA from chimeric quail embryos at day-7 by PCR.

3.3 Detection of chicken DNA in semen of adult quail

Four quails were hatched from 68 eggs injected with chicken GCR cells. They were 2 males and 2 females after being raised to sexual maturity. Chicken DNA was detected from the semen of only one male. The intensity of the PCR signal was estimated at least 1/100 comparing with the positive control.

The sequence of amino acid is longer in chicken than in quail ovalbumin. In designing the primer set in the present study, this difference has been considering. The primer set for chicken ovalbumin was able to amplify PCR products with chicken, but not quail genomic DNA. Other available primer sets to detect interspecific chimera between quail and chicken have been reported [6, 7]. One of them is microsatellite locus LE10171 on the chicken Z chromosome (GenBank #X85538) and another, of which the original gene is unknown, amplifies PCR products in both chicken and quail DNA templates with different sizes (923 bp and 458 bp, respectively). As well, the peafowl-specific primers designed from cyt b gene sequences have been used to detect the interspecific chimera between peafowl and chicken [27].

The donor chicken DNA was detected in the gonad of the recipient quail embryo and in the semen of the recipient male quail as revealed by PCR. The CD cells, including pre-PGCs in blastoderm at stage X [15,17-23] as the donor, showed the possibility to produce the interspecific germline chimera. However, its efficiency was very low in the present study. The cells of GCR, including PGCs at stage 7-8 [16,24-26], having been expected to have the possibility to produce the interspecific germline chimera, also showed very low efficiency. Low survival rate of embryos injected with GCR cells is a critical problem. It seems to be an exceptional occasion that the interspecific germline chimera quail is obtained in the present study. Further studies are needed to make necessary improvement.

The transfer of PGCs collected from the blood at stage 13-16 [24] is the most efficient method to produce the interspecific germline chimera [2-4, 6, 7, 28, 29]. Li et al [7] reported that the chicken genome-specific PCR product was observed in the semen of adult quail using circulating PGCs of chicken as the donor. However, it needs a complicated protocol to utilize the circulating PGCs. Methods to facilitate the utilization of PGCs are desired.

In order to help producing the germline chimera, the recipient embryos were treated with cytotoxic drug as busulfan [9, 30], exposed to gamma-rays [31] and soft X-rays [7,32], removed a center cell cluster from the central disk [23], or bled [3, 4] prior to the injection of donor cells. However, special chemicals, instruments or skills are necessary for these pretreatments.

The simplest method to produce the interspecific germline chimera was used in the present study. However, there are several critical problems, e.g., the low survival rate of embryos at day-7 and the low hatching rate; in addition, the method of employing CD and GCR cells are far from efficient.

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Correspondence to: Dr. Tomoki Soh, Laboratory of Reproductive Physiology and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan.
Tel +81-92-642 2940, Fax +81-92-642 2938
E-mail: soht@agr.kyushu-u.ac.jp
Received 2001-05-31      Accepted 2002-06-04