Macromolecule BAC targeting vector for transfection of chicken embryo primordial germ cells
- Dong-sheng, Tang
- Jin-rong, Liu
- Hong, Jiang
- Fang, Zeng
- Dao-yuan, Gong
- Zhen-dan, Shi
- Xi-quan, Zhang
Abstract
BACKGROUND:
Using the primordial germ cells (PGCs) technology, the foreign genes can be easily transferred into the chicken to get the transgenic chicken in shorter time. However, till now, the transfection efficiency of PGCs is very low and it is specially difficult that longer gene fragment is transfected into PGCs.
OBJECTIVE:
To achieve the transfection of exogenous gene of macromolecule BAC vector into PGCs.
DESIGN, TIME AND SETTING:
The cytology in vitro study was performed at the Foshan University from August 2006 to July 2008.
MATERIALS:
Hatching egg was obtained from Yuehuang chicken at the chicken farm of Southern China Agriculture University. Fertile egg was hatched at 37.5 °C under relative humidity of 65% for 68-72 hours. BAC-TDN-GID vector was maintained at the Laboratory of Molecular Biology, Foshan University.
METHODS:
Using the DNA sequences of rRNA repeating gene family and its internal transcribed spacers as homologous recombination directed sequences (HRDS), the vector of macromolecule BAC containing GFP gene and hIFN gene had been constructed. The constructed vector containing GFP gene and hIFN gene was identified and then the positive clone would be expanded cultured and extracted on a large scale. The genital ridge or the gonad of stage 19 embryos were picked and got. PGCs were got by digestion with trypsin. PGCs were incubated on feeder layer cells with growth factors. The vector was transferred into PGCs by using the liposome mediate method. The survival cells were identified in the levels of DNA and RNA.
RESULTS:
The PGCs from the genital ridge or the gonad of stage 19 embryos were mulberry-like shape or oval at 48 hours. Cell colony was formed and prunosus was found following periodic acid-Schiff staining at 72 hours. The BAC-TDN-GID of macromolecule vector whose molecular weight is 30 kb was successfully transferred into PGCs by the existence of fluorescence cells. Polymerase chain reaction and reverse transcription-polymerase chain reaction had confirmed that the fragment of amplified products was identical to expecting results. Target gene hIFN had been inserted into the genome of PGCs and expressed.
CONCLUSION:
The exogenous gene on the macromolecule BAC vector has been transferred into in vitro cultured PGCs.
Figure
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