Construction of the subtractive cDNA library of injured adult and fetal rabbit skins.
OBJECTIVE
Early gestational mammalian fetuses possess the amazing ability to heal cutaneous wounds in a scarless fashion. Over the past years, scientists have been working to decipher the mechanisms underlying this regenerative repair. The remarkable phenotypic differences between fetal and adult healings behooves us to learn their characteristics in genetics, which represents potentially important mechanisms involved in wound repair observed in fetal versus adult tissues. In this sense, it is reasonable to construct subtractive cDNA library for future research.


METHODS
Middle laparotomy and hysterotomy were performed on pregnant rabbits at 20-day gestation to expose the fetal back, and a longitudinal incision through the skin was made on the back of the fetus. The traumatized fetal skin was harvested 12 hours post-operation, the fetus control and traumatized adult skin specimens were taken at the same time. dscDNA was synthesized from total RNA of skin samples with SMART technology. Taking one of the three samples as Tester respectively and the other two as Drivers, we obtained 1 forward and 2 reverse hybridization products. After being amplified with selective polymerase chain reaction, the products were inserted into a vector, and then transferred into E. coli HB101. The colonies were screened afterwards.


RESULTS
The wounded fetuses were alive for a long time even after birth. Every determinant step, such as RNA isolation, cDNA synthesis, Rsa I digestion, adaptor ligation and hybridization, was well-operated. Subtractive efficiency identification demonstrated that the suppression subtractive hybridization (SSH) was successful. Insertion into vector and transferring to E. coli were satisfactory.


CONCLUSIONS
Instead of classic SSH, an improved SSH with 2 Drivers was applied for the experiment. Results confirmed that the improved program was reasonable and correct in both theory and practice. The subtractive cDNA library we have obtained is going to be used for future researches to reveal scarless healing related gene(s) and its (their) expression.
Zhang
Bo
B
Research Institute of Surgery, Daping Hospital, Chongqing 400042, China. john_power1201@yahoo.com
Liu
Da-wei
DW
Wang
Zheng-guo
ZG
Zhu
Pei-fang
PF
Zhou
Ji-hong
JH
Jiang
Jian-xin
JX
D000818:Animals; D002921:Cicatrix / Q000235:genetics*; D018076:DNA, Complementary; D004195:Disease Models, Animal; D005260:Female; D005333:Fetus; D015723:Gene Library; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011270:Pregnancy, Animal; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D012680:Sensitivity and Specificity; D012867:Skin / Q000293:injuries*; D014945:Wound Healing / Q000235:genetics* / Q000502:physiology
D018076:DNA, Complementary; D012333:RNA, Messenger