Establishment And Biological Characteristics Of Fibroblast Cell Bank In Seven Goat And Sheep Breeds

Taihangshan Black Goat ,Jining Grey Goat, Dorset Sheep, Texel Sheep, Suffolk Sheep and Angora Goat were selected as experiment materials.By means of tissue block culture, we cultivated the ear margin tissues in vitro. When subcultured several times, the fibroblasts were collected and frozen in liquid nitrogen, now the cell banks were established successfully.Their growth dynamics,genetic stability and any kind of contamination were studied such as the growth curve, karyotype analysis ,isoenzyme bands analysis and transfection by Fluorsent Protein Vectors etc. our aim is to obtain an effective way to coserve and resort the animal genetic resources at cell level, furthermore to provide some references for others. The cell freezen amount is about 3×10~6 cell/ml, sample contents of each breed were between 30 and 60, cryopreservation ampules were 150-180 each breed. The experiment results were as follows:1. The cultured cells were typical fibroblast morpha.which growed as irregular triangle or shuttle , appearing as radioactive blaze and helix in shape.2. Their growth curve were "S"which showed the population double time (PDT)were 24 hours since the second day, the cells understood latent phase, logarithmic growth phase and stagnate phase.3. The bank cells could be sucessfuly recovered for many times.Though affected by many experiment factors during cryopreservation ,with typan staining the recovered cell's survival ratio up to over 90 (?). Adherent cell after revival growed as fast as cell before freezing. Meantime the fibroblasts became more purified.4. the fibroblasts identified with Microbial detection test were negative ,which demonstrated cultured cells were not contaminated by bacteria,yeasts,fungi,virus and mycoplasmas.5. The number of chromosome is 2n=60 in Black Goat, Jining Grey Goat, Angora goat and 2n=54 in Dorset Sheep, Texel Sheep, Suffolk Sheep.Results of chromosomal karyotypes indicated that cells are diploid,and the hereditary character is stable.chromosome character can identify cell lines.6. Electrophoretic Analysis of isoenzyme banding patterns were as follows: there were 3 bands of LDH in each breeds ,the rate of relative migration is identical, the relative migration sequences were LDH1, LDH2, LDH3 from "+"to"-". There are 2 zone bands of MDH in seven breeds, the sMDH went fast then mMDH.7.Transfecting fibroblasts with Fluorescent Protein Vectors mediated by lipofectin.The test indicated that pEGFP-N3 plasmid could be effectively transfered into fibroblasts by lipofectin .the transfection conditions involved fibroblast growth state, DNA/lipofectin mixing concentration ratio,and exposure time of fibroblast to DNA lipofectin complexes. The highest efficiency was achieved 60% and 80%.the essential transfection conditions for these efficiencies were in coating pEGFP-N3 of 2.0μgDNA with lipofectin8μl,and exposing the fibroblasts to the DNA-lipofectin complexes for 48 hours . The transfecting efficiency in the fibroblasts of Dorset sheep is 20%.By Constructing the animal cell bands,we can conserve the genetic resources of domestic animal permanently at cell level,and provide the valuble conservated materials for other biological studies.