分子生物学课件-基因打靶和遗传修饰动物模型

1. Vector construction
Neomycin belongs to aminoglycoside class of antibiotics that contain two or more aminosugars connected by glycosidic bonds.
1. Vector construction
The BIG Breakthroughs
到1987年，T。
到目前為止，運用基因同源重組、非同源末端 連接（non- homologous end-joining, NHEJ）或 同源 指導修復（homology-directed repair, HDR ）進 行基因敲除（替換）依然是構建基因敲除 （遺傳 修飾）動物模型中最普遍的使用方法。
Non-homologous DNA Flanked by Homologous DNA
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3. Homologous recombination
❖Cre/loxP System or Cre-Lox recombination
Cre (Causes Recombination) ; loxP (locus of crossing (x) over, P1)
- results in: - endogenous DNA replaced with exogenous - specific - targeted - copy number = 1
3. Homologous recombination
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3. Homologous recombination
TALEN Clustered Regulatory Interspaced Short Palindromic
Repeats (CRISPR) sequences with CRISPR-Associated Protein 9 (Cas9) , CRISPR/Cas9 The NgAgo–gDNA system Structure guide endonuclease (SNG) system
Cells are pluripotent Able to differentiate into many (all?)
different cell types in embryo.
2. Embryonic Stem Cell Isolation
❖Isolated from a pre-implantation embryos
基因打靶和遺傳修飾動物模型
The Gene Targeting and Heredity-Modified Animal Model
Introduction
❖Gene Targeting (Knock out)
Homologous recombination Zinc Finger Nuclease, ZFN Transcription-Activator-Like Effector Nuclease ,
Homologus Recombination
Homologous recombination with transfected disruption constructs can inactivate specific target genes in ES cells. When recipient diploid cells are transformed with the gene disruption construct, homologous recombination between the ends of the construct and the corresponding chromosomal sequences will integrate gene into the chromosome, replacing the target gene sequence.
TALEN、CRISPR/Cas9、The NgAgo–gDNA system ，它們同樣可以達到基因敲除的目的。
The BIG Breakthroughs
Discovery that ES cells derived from preimplantation embryos (blastocysts) were pluripotent (Martin Evans in 1981).
實現基因敲除的多種原理和方法
利利用基因因同源重重組組進行行基基因敲敲除 除
條條件件性性基基因因敲敲除除法 法 利利用用隨隨機機插插入入突突變變進進行行基基因因敲敲除 除 RRNNAA interference，RNAi引起的基因敲除除。。
誘誘導導性性基因敲除法 法 基基因因捕獲法 法
ZZFFNN,, TTAALLEENN,, CCRRIISSPPRR//CCaass99,, NNggAAggoo––ggDDNNAA ssyysstteemm,, SSNNGG ssyysstteemm
Introduction
❖Transgenetic technology (Knock in)
random integrations
Introduction
❖Gene targeting in mouse embryonic stem cells is now a well-established technique that is widely used to create animal models for human disease or to study gene function at the level of the whole animal.
2. Embryonic Stem Cell Isolation
❖Isolated from a pre-implantation embryos
➢ From inner cell mass of blastula stage embryo ➢ Cells are undifferentiated
3. Homologous recombination
3. Homologous recombination
❖Gene Targeting Technology
Cells take up exogenous DNA ➢ Frequency is very low ➢ Can be induced to higher frequency (chemical, electrical, injection)
概述
基因敲除是自80年代末以來發展起來的一種新型 分子生物學技術，是通過一定的途徑使機體特定的 基因失活或缺失的技術。
通常意義上的基因敲除主要是應用DNA同源重 組原理，用設計的同源片段替代靶基因片段，從 而 達到基因敲除的目的。
概述
❖ 隨著基因敲除技術的發展，除了同源重組外，新 的原理和技術也逐漸被應用，比較成功的有ZFN、
In Dividing Cells: ➢ Some DNA incorporated into genome
Random integration — rare event Homologous Recombination — even more rare
3. Homologous recombination
表型研究 得得到到純純合合體 體
1. Vector construction
Neomycin R
Neomycin R
Construct for disrupting a target gene can be prepared by the PCR. The two primers designed for this purpose each contain a sequence of about 20 nucleotides (nt) that is homologous to one end of the target yeast gene as well as sequences needed to amplify a segment of DNA carrying a selectable marker gene such as Neomycin R, which confers resistance to G-418.
3. Homologous recombination
- driven by the DNA sequences (targeted) - rare (1000× less frequent than random) - increased efficiency: - isogenic DNA - longer stretches of homology
Homologous recombination with transfected disruption constructs can inactivate specific target genes in cells.
利用同源重組構建基因敲除動物模型的基本步驟
基因載體的構建 ES 細胞的獲得
同源重組 選擇篩選已擊中的細胞
同源重組
同源重組（Homologus Recombination） 是指發生 在非姐妹染色單體（sister chromatin） 之間或同一染色 體 上含有同源序列的DNA分子之間或分子之內的重新 組合 。同源重組需要一系列的蛋白質催化，如原核生 物細胞 內的RecA、RecBCD、RecF、RecO、RecR等 ；以及真 核生物細胞內的Rad51、Mre11-Rad50等等。
➢ grown in culture need cells to divide but not differentiate longer time in culture = more differentiation.
2. Embryonic Stem Cell Isolation
Inner Cell Mass
Cre Recombinase is a tyrosine recombinase enzyme derived from the P1 Bacteriophage. The enzyme uses a topoisomerase I like mechanism to carry out site specific recombination events.
1. Vector construction
❖Resistance to G418 is conferred by the neo gene from Tn5 encoding an aminoglycoside 3'phosphotransferase, APT 3' II.
❖The bacterial neomycin phosphotransferase (neo) gene.
❖Random Integration:
DNA incorporated anywhere in genome (not targeted)
copy number can be very high. disrupts the endogenous DNA at insertion site. transgenic animals are usually random integrations.
G418 (Geneticin) is an aminoglycoside antibiotic. G418 blocks polypeptide synthesis by inhibiting the elongation step in both prokaryotic and eukaryotic cells.
Discovery that mammalian cells in culture could undergo homologous recombination with genetically engineered DNA (Mario Capecchi and Oliver Smithies in the 1980’s).