Basic strategy of gene function analysis

1. Basic strategy of gene function analysis

3. Section 1. transgenic model • 组织及动物整体水平转基因获得的转基因动物 Transgenic models obtained on Tissue and whole animal • 细胞水平转基因获得的基因转染细胞模型 Transgenic cell models obtained on cellular level

4. 1、transgenic animals • Transgenic technology: Exogenous gene is introduced into fertilized egg cell or embryonic stem cell and recombined randomly on chromosome, resulting in that exogenous gene is inserted into genome of recipient cell and pass on to the next generation. • Transgenic animal： exogenous gene is introduced and integrated into genome by manipulative methods and obtain the animal harbored the gene. feature: to operate on molecular or cellular level, to express on tissue or whole animal level.

5. Basic process preparing transgenic animal • The vectors carrying the desired gene are inducted into the fertilized eggs or embryonic cells with microinjection or other methods. • The fertilized egg or embryonic cell of containing a desired gene is imbedded into oviduct to develop transgenic animal, • The relation between the gene and the phenotype of animal is analyzed to understand the function of the desired gene.

6. The technique of gene introduction: physic, chemical and biologic methods. 1) microinjection 2) embryonic stem cells (ES) 3) retroviral infection 4) Spermatozoa bearer

7. microinjection

8. Separating and culture of embryonic stem cell Embryonic stem cell Blastodermic vesicle Separating Endothecium cell Cell culture

9. basic process preparing transgenic animal

11. Application of transgenic animal • 研究基因表达的组织或阶段特异性 • 通过研究转入外源基因后的新表型，可以发现基因的新功能 • 外源基因的随机插入，可能导致某内源基因突变，分析这些突变表型，可发现新基因 • 用于只在胚胎期才表达的基因的结构和功能的研究 • 建立研究外源基因表达、调控的动物模型 • 研究遗传性疾病 • 建立人类疾病的动物模型，为人类的基因治疗提供依据 • 动物新品种的培育 • 基因产品的制备 • 用于对免疫机制、免疫相关疾病的研究及建立免疫性疾病动物模型

12. 2、cell models of gene transfection • to clone a desired gene into eukaryotic expression vector → to transfect cell → to obtain the cell models ofgene over-expression.

13. basic process • Separation and purification of desired gene • To clone the gene to expressive vector • To transfect the recombinant vector into cell

14. Separation and purification of desired gene

15. To clone the gene to expressive vector

16. Transfection of the recombinant vector into cell

18. Section 2. Gene knock-out • to alter a specific gene by fixed–site homologous recombination and research the gene function in vivo. • gene knockout：fixed-site specific gene knockout • gene knockin: fixed-site substitution of a desired gene for a specific gene in genome.

19. 1、essential condition of gene targeting • Embryonic stem cell (ES) Feature: ES got from the fourth and fifth day from fertilized egg to Blastodermic vesicle. （1）Culture in vitro, remaining totipotency of development （2）The morphology of adhesive growth cell in vitro: big nucleus, less kytoplasm, dense alignment, settlement growth. （3）When poor differentiation condition, many Functional genes are non-expression and cell proliferated and differentiated genes are expression. When culture in vitro, ES possesses propensity to differentiate to various tissue.

20. Targeting vector containing two screening mark： （1）neo positive screening mark: neo gene is linked between two homogeneous arm of exogenous gene. As homogeneous recombination, neo gene is inserted into chromosome and the cells can grow in the medium containing G418. （2）HSV-tk negative screening mark: As non-homogeneous recombination, random integration of HSV-tk gene, the coding product breakdown nucleotide to poison and cell death.

21. construction of targeting vector Plasmid vector Homologous gene fragment HB1 HB2 Neor gene HSV-tk gene

22. 2、basic process of gene knockout • Construction of targeting vector Basic process： ① homogeneous fragment (to treat gene) of desired gene cloning into vector. ② to cut the most part of homogeneous sequence, remaining a small part of sequence on two end of linear vector. ③ neo gene cloned between tow homogenous arm and HSV-tk cloned on a end of the recombinant vector.

23. Introduction of targeting vector into ES cell with the ES cell, a specific gene is substituted by homogenous recombination of targeting vector with the knock-outing gene. • Injecting the ES cell into blastodermic vesicle the ES cells of gene knockout form cell clump with the normal cell in blastodermic vesicle. • Embedding blastodermic vesicle in womb of pseudocyesis mouse. tow kinds ES cells in the blastodermic vesicle: the normal ES cells——developing normal mice. the ES cells of gene knockout——developing the mice of gene knockout. • chimeric Crossbred mice screening chimeric mice of gene knockout.

24. Constructing mouse model of gene knockout

26. 3、conditional gene knockout • Because Cre recombinase can recognize and cut sequence LoxP (34bp) for achieving precise genetic manipulation in mice. Many of these desired genetic manipulations rely on Cre's ability to direct spatially and temporally specified excision of a pre-designated DNA sequence that has been flanked by directly repeated copies of the loxP recombination site.

27. The vector of conditional gene knockout

28. The vector of conditional gene knockout

29. The CKO constructs generated in the GTTF contain:

31. Application of Gene targeting on medicine • To construct the animal model with human diseases. 1) genetic disorder 2) tumor 3) gene therapy • To identify new genes in human body or animal and their function.

32. Section 3 gene suppression and gene silencing • Antisense RNA • RNA interference

33. Antisense RNA • 能与mRNA互补配对的RNA分子。因其在空间上妨碍mRNA为模版的蛋白质合成，可在翻译水平调控基因表达。 • Antisense RNA (aRNA) is single-stranded RNA that is complementary to a mRNA strand transcribed within a cell. That regulates the gene expression on the translation level.

34. Mechanism of antisense RNA

36. 反义RNA的作用 • 根据碱基互补原理，用人工合成或生物体的特定互补DNA或RNA片段抑制或封闭基因表达的技术。 • 在RNA水平上分析特定基因的可能功能 • 可以设计与疾病（癌）基因或相应mRNA互补的反义DNA或RNA片段，封闭癌基因。

37. RNA interference (RNAi) • Short double-stranded RNA (dsRNA) is used to induct degradation of homogenous mRNA --The ability of dsRNA to suppress the expression of a gene corresponding to its own sequence is called RNA interference (RNAi). It is also called post-transcriptional gene silencing (PTGS)

38. Mechanism of RNAi

39. Normal condition in cells

40. Basic process of RNAi • Screening RNAi site to a target mRNA • Synthesis of siRNA corresponding to atarget mRNA • Construction of recombinant vector carrying a siRNA • Transfecting cell with recombinant vector • Analysis of target mRNA and it’s protein

41. RNAi basic process