Pcgf2 promotes the reprogramming of chicken embryonic fibroblasts into induced pluripotent stem cells
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摘要:目的
以提高诱导鸡多能干细胞的干性为目的，利用Pcgf2基因与传统诱导多能干细胞的重编程因子共同诱导鸡多能干细胞。
方法以鸡早期胚胎为模板，对Oct4、Sox2、Nanog、Lin28、C-myc、Klf4这6种传统的重编程因子及Pcgf2进行基因扩增，并与慢病毒载体重组为慢病毒质粒，在包装慢病毒并测定相对应的病毒滴度后，分组感染鸡胚成纤维细胞，在诱导的过程中对干细胞标记物进行检测。
结果利用传统的重编程因子组合与加入Pcgf2共同诱导后的细胞都较早地表达出磷酸酶活性。加入Pcgf2诱导多能干细胞可以使细胞更早地出现类干细胞形态，且内源性基因Oct4、Sox2、Nanog和Lin28的表达得到更稳定的提高。
结论Pcgf2在加入诱导体系后可以有效提高细胞的重编程接近干细胞状态。该研究可为选择重编程因子诱导鸡细胞完全重编程及其机制研究提供理论参考。
Abstract:ObjectiveWe aimed to improve the stemness of chicken induced pluripotent stem cells using the Pcgf2 gene together with conventional reprogramming factors to induce chicken pluripotent stem cells.
MethodSix conventional reprogramming factors including Oct4, Sox2, Nanog, Lin28, C-myc and Klf4 as well as Pcgf2 gene were amplified and reconstituted with lentiviral vectors as lentiviral plasmids using chicken early embryos as templates. After packaging the lentivirus and measuring the corresponding viral titers, chicken embryonic fibroblasts were infected in groups and stem cell markers were assayed during the induction process.
ResultThe cells induced by the combination of conventional reprogramming factors and Pcgf2 all expressed phosphatase activity early. Inducing the pluripotent stem cells with Pcgf2 could make the cells appear stem-like morphology earlier, and the expressions of endogenous genes Oct4, Sox2, Nanog and Lin28 increased more stably.
ConclusionPcgf2 can effectively enhance the reprogramming of cells to approach the stem cell state after being added to the induction system. The research provides a theoretical reference for the selection of reprogramming factors to induce complete reprogramming of cells in chickens and the mechanism study.
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图 1 不同多能基因的PCR结果
Figure 1. PCR results of different pluripotent genes
M：DL2000 DNA marker
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图 2 不同多能基因的重组慢病毒质粒酶切结果
Figure 2. Enzyme digestion results of recombinant lentiviral plasmids with different pluripotent genes
M：DL15000 DNA marker
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图 3 倒置荧光显微镜下观察293T包装慢病毒
Figure 3. 293T packaging lentivirus observed under inverted fluorescence microscope
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图 4 倒置荧光显微镜下观察倍比稀释后细胞感染慢病毒情况
Figure 4. Cells infected with lentivirus after multiplier dilution and observed under inverted fluorescence microscope
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图 5 碱性磷酸酶活性检测结果
A：Lv-OSKMNL；B：Lv-OSKMNL+Pcgf2；C：空白对照 Mock
Figure 5. Results of alkaline phosphatase activity assay
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图 6 荧光镜检观察重编程细胞聚团形成过程
Figure 6. Fluorescence microscopy observation of the formation process of reprogrammed cell clusters
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图 7 不同内源性基因的荧光定量PCR检测
各图中，相同时间柱子上方的不同小写字母表示处理间差异显著(P<0.05， LSD法)
Figure 7. Fluorescence quantitative PCR assay of different endogenous genes
In each figure, different lowercase letters above columns at the same time indicate significant differences among treatments (P<0.05, LSD method)
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图 8 三胚层标记物的荧光定量PCR检测
各图中，相同时间柱子上方的不同小写字母表示处理间差异显著(P<0.05， LSD法)
Figure 8. Fluorescence quantitative PCR assay of triploblastic markers
In each figure, different lowercase letters above columns at the same time indicate significant differences among treatments (P<0.05, LSD method)
表 1 多能基因编码区序列的PCR同源重组扩增引物
Table 1 PCR homologous recombinant amplification primers for the coding region sequences of the pluripotent genes
基因
Gene正向引物序列 (5′→3′)
Forward primer sequence反向引物序列 (5′→3′)
Reverse primer sequence产物大小/bp
Product sizepCDH-CMV GGTGGCTAGCTCTAGAATCTTCTATGGAGGTCAAAACAG GGATCCGCGGCCGCAAGGATCTGCGATCGCTCCGGT 8176 Oct4 GGCACCGGAGCGATCGCAGATCCTTGCGGCCGCGGATCCTCAGTGGCTGCTGTTGTTCA CTGTTTTGACCTCCATAGAAGATTCTAGAGCTAGCCACCATGCATGTAAAAGCCAAAAA 888 Sox2 GGCACCGGAGCGATCGCAGATCCTTGCGGCCGCGGATCCTTACATATGTGATAGAGGGA CTGTTTTGACCTCCATAGAAGATTCTAGAGCTAGCCACCATGTACAACATGATGGAAAC 968 Nanog CTGTTTTGACCTCCATAGAAGATTCTAGAGCTAGCCACCATGAGCGCTCACCTGGCCAT CTGTTTTGACCTCCATAGAAGATTCTAGAGCTAGCCACCATGAGCGCTCACCTGGCCAT 930 Lin28 GGCACCGGAGCGATCGCAGATCCTTGCGGCCGCGGATCCTCATTCCCGGGTTTCGGGGC CTGTTTTGACCTCCATAGAAGATTCTAGAGCTAGCCACCATGGGGTCTGTTTCCAACCA 609 C-myc TCGCAGATCCTTGCGGCCGCGGATCCCTATGCACGAGAGTTCCTTAG TTTTGACCTCCATAGAAGATTCTAGAGCTAGCCACCATGCCGCTCAGCGCCAGCCT 1251 Klf4 TCGCAGATCCTTGCGGCCGCGGATCCTTAAAAGTGCCTCTTCATGT TTTTGACCTCCATAGAAGATTCTAGAGCTAGCCACCATGCGGCAGCCCCCCGGCGA 1377 Pcgf2 GGAGCGATCGCAGATCCTTGCGGCCGCGGATCCTTAGGTCAGCGCGGACCCC TGACCTCCATAGAAGATTCTAGAGCTAGCCACCATGCACAGGACCACCCGGATA 966 
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表 2 干细胞标记物荧光定量PCR检测引物
Table 2 Primers for fluorescent quantitative PCR detection of stem cell markers
基因名称
Gene name正向引物序列(5′→3′)
Forward primer sequence反向引物序列(5′→3′)
Reverse primer sequenceEndo-Oct4 CTGGCCCCAGGCAGGTAA CGGGATCTCCATGAACAACAG Endo-Sox2 AAAAGGTCCAGAATTTCTAATA CCCCAAGCAGACTTCATA Endo-Nanog TAGTAGTGTCCGCACCTAAC GTATGCAACCAGCTCACC Endo-Lin28 CAAACAAACCCAAAGATACG AAAGCCAATGCCAAGTGA Sall4 GAACTTCGGTCGTGGCACAAGG AATACCAAGTGAGCGTCCCATTAGC Gata4 ACAGTTGACACATTCTCGCCCTTC CCACTTGGACTTCTTCGCCCTTC Gata6 ACACCAGTGATCCTGCCTGACG AGAGAGCACCAGTCCCGAAAGC Pax6 ATGGGCTGGCTATTCATG CCCCGTTTCCTCTTTCAC GAPDH GCACGATGCATTGCTGACAA GGGTGGTGCTAAGCGTGTTA
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