Studies On Production Of Transgenic Goat By Somatic Cell Nuclear Transfer

Transgenic animal research is one of the most active areas in biology, which can changethe genctic component and productive performance in boma1 according to mankind mind.The Production cfficicncy of transgenic bomal by the traditional method of pronucleusmicroinjection is very low (The efficiency of transgene is about 3~5%.). ln comparison withthe traditional method, this technique of somatic cell nuclear transfcr has many advantageswhen used in transgenic animal production. The main advantae is that transgcnic procedurecan be operated in somatic ccll in vitro. which leads to grC8t imProvcment of productiveefficienCy and decrease of productive cost. lt's easy to get a lot trAnsgenic animals with highlexel exPress1on of fOreign gene, because the transgenic expression could be controlledartificlall}-. The stUdies were apaplied in the field of modificaton of gene on the special locus.the biological medicines, the gene function researchs and so on. The Myostatin gene locuswas selected for the gene targetlng in this stud3'. The transgenic fram vector was consthected,which contains neo' gene and GFP gene as positive selection markers, TK gene as negtivemarkef, loxP locus as dcleting site of Cre enZyme. Using it for basic frame consmict, fetalfibroblast cell and aged fibroblast cell in goats were transferred. The transfection efficiency ofdifferent methods were determine, and many positivc transgenic cell clones and sometransgenic embryos were produced. We have built a set of Perfect protocoI making transgenicsmicture. passage culture of somatic cell, the identification of the sex, transgene, seIection oftransgenic cell lines, and the production of transgenic nuclear transfer embryos, which willPrdride a dsve experiInental method for makin transgemc somatic celI cloning goat.l. Fetal Fibroblast cells were isolated from seven 25~35-day old goat fetUs afer prcgnancy.7 sometic cell lines were produced after passage cultured in vitro. The sex of these seven ceIllines were tested b}' PCR reaction, 4 cell lines are female and the other are male. The result ofchromosome ploidy analysis to thTee cell lines (3 female), indicated that the ploidy of thesecell lines maintained normal when cultUred up to passage 20. 75W84.44% cultUral cellsmaitain the normal ploidy of chrmosome.2. According to the published DNA sequence of Myostatin gene in sheep. pig, ndce andbovine. Wc designed tWo pairs of primers, which contain speCific enZyme-degesting locus atthe 5' of each primer to guarantee the oried ligation in the vector. It's the first hme to haveclOned the complet0 DNA sequence of intron I and II of MyOStatin gene, and which werepublished in thc data base of intemational Genbank. Thc accession numbcr of intron I and IIwere; am93639, AY032689, in goat and AF393618, AF266758 in shopWly3. Using the long fragment PCR method. the laree 4.5kb boent chchg from cxon Ito III as 5' ann and l.4 kb from exon III tO 3' UTR as 3' arm in the hageulc veeor.Aner analysis of sequence and homology of such fmpmentS, they were correct, and thehomolOgouse recombinant constrUctUre was fmisked, which was named as pLOxP-M. It willbecome lose 57l bp DNA scquence and l l9 amino acid betWeen tWo bomolOgouse arms,l112002.11and make frameshift and code mutation, which cause the lose of activity in myostatin gene, resulting to double muscle phenomena of transgenic animal.4. Using linear GFP to transfecte fetal fibroblast in goat with different methods (Ca-phosphate. lipofectamine-mediated, electric pulse-mediated ), it's found that 〥NA concentration should be 2-4 u g and the time should be 8~10h for Ca-phosphate method, @ DNA concentration was 2 n g, transfer time was lOh, lipofectamine 8 1J L for lipofectamine-mediated method; (3)the formula was 0.6kv/cm DC pulse and lasting time was 15 ms for electric pulse-mediated method, which could resulte in a high transfection efficiency.5. 800 y g/mL G418 was used as working concentration, after cell toxicity test of G418. In experiment, it was selected fo...