本論文分為二部份，第一部份研究的目的在選殖白色來亨雞之端粒反轉錄酶 (telomerase reverse transcriptase, TERT) 基因並進行 DNA序列分析比對，期能供基因轉殖並發展延長家禽體細胞體外培養的技術平台。試驗應用顯微抽取技術自雞蛋取得原腸期雞胚供萃取 RNA樣品，經過反轉錄-聚合酶連鎖反應(reverse transcription-polymerase chain reaction, RT-PCR) 合成互補 DNA (complementary DNA, cDNA)，並設計特異引子進行 PCR選殖雞的 TERT。結果 PCR之後獲得一 4.7 kb的產物，此產物經膠體回收並選殖入TOPO載體，經過 DNA序列分析與 Basic Local Alignment Search Tool (BLAST) 比對，確定為雞的 TERT基因。第二部份是分析行政院農業委員會畜產試驗所應用體細胞核轉置 (somatic cell nuclear transfer, SCNT) 技術生產的複製荷蘭種乳牛與阿爾拜因乳山羊及其後代動物的端粒 (telomere) 長度。將複製動物的端粒長度與同年齡的非複製動物進行比較，以了解複製與非複製動物及其後代的端粒長度變化。試驗自複製牛與羊採集全血，經離心分離白血球供萃取基因組 DNA，然後應用套組測定端粒限制片段長度（Telomere Restriction Fragment, TRF）。結果顯示，年齡在 2歲至 3歲間的 4頭複製乳牛之端粒長度與同年齡非複製牛之端粒長度相近。複製牛的 4頭後代之端粒長度也與其同齡非複製乳牛相似；顯示複製牛及其後代之端粒長度與年齡相近之非複製乳牛之間並無差異顯著（p＞ 0.05）。而3頭月齡不同的複製羊，其端粒長度則較同齡之非複製對照羊為短，並隨著月齡的增加，端粒長度顯著較非複製對照羊為短 (p＜0.05)。而複製羊的 5頭後代之端粒長度則與同齡對照羊隻相近而無差異顯著（p＞0.05）。本研究顯示，應用複製技術生產的乳牛，其端粒長度在經過 NT操作後，胚在發育過程其基因組順利地經過再程式化，使端粒長度回復正常，並未因為應用成體供核體細胞進行複製而導致端粒短化。而應用 SCNT複製羊其端粒長度較對照正常羊為短，且隨著月齡增加而與對照之正常羊隻為短，達到統計上的顯著差異結果，此結果顯示不同複製動物之端粒長度變化並不一致；而複製動物後代的端粒長度，則與正常動物相似。 This thesis consists of two parts. The objective of the first part was to clone and sequence the telomerase reverse transcriptase gene (TERT) from gastrula stage embryos of White Leghorn chicken. Total RNA were first extracted by using commercial kit and followed by RT-PCR with specific primers to obtain the full-length of putative TERT cDNA. The obtained PCR product was recovered by agarose gels electrophoresis and then subcloned into TOPO vectors for DNA sequencing. BLAST comparison of the cloned DNA sequences with NCBI Genomes database confirmed the identity of chicken TERT (chTERT). The results showed that the chTERT gene had been successfully cloned and could be subsequently used for gene transfer to establish long-term in vitro culture system for avian cells.
The objective of the second part was to compare the telomere length of somatic cell nuclear transfer (SCNT) cloned animals and their offspring with the non-cloned counterparts. Genomic DNA for terminal restriction fragment (TRF) analysis was purified from whole blood of cloned animals, their offspring and age-matched control animals. The results showed that there was no significant difference in the telomere lengths between cloned cattles and their age-match cattles aged 2-3 and 3-4 (p＞0.05). Similarly, the average telomere lengths of the offspring of cloned cattles were not significantly different from their age-matched counterparts. However, the cloned dairy goats at different ages have shortened telomere lengths when compared with age-matched controls. Offspring derived from cloned goats had normal telomere lengths compared with their age-matched counterparts. Our results suggested that the telomere lengths of cloned animals were varied among species. The telomere lengthes in cloned goats were not completely restored as those in the cloned cattle by SCNT from chicken.
