Basic strategy of gene
function analysis
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
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.
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.
The technique of gene introduction:
physic, chemical and biologic methods.
1) microinjection
2) embryonic stem cells (ES)
3) retroviral infection
4) Spermatozoa bearer
microinjection
Separating and culture of embryonic
stem cell
Blastodermic
vesicle
Separating
Endothecium cell
Embryonic
stem cell
Cell culture
basic process preparing transgenic
animal
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
2、cell models of gene transfection
 to clone a desired gene into eukaryotic
expression vector → to transfect cell
→ to obtain the cell models of gene
over-expression.
basic process
 Separation and purification of desired
gene
 To clone the gene to expressive vector
 To transfect the recombinant vector
into cell
Separation and purification of desired gene
To clone the gene to expressive vector
Transfection of the recombinant vector into
cell
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.
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.
 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.
construction of targeting vector
Plasmid
vector
Homologous
gene fragment
HB1
HB2
Neor gene
HSV-tk gene
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.
 Introduction of targeting vector into ES cell
with the ES cell, a specific gene is substituted by
homogenous recombination of targeting vector with the knockouting 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.
Constructing mouse model of gene knockout
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.
The vector of conditional gene knockout
The vector of conditional gene knockout
The CKO constructs generated in the GTTF contain:
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.
Section 3
gene suppression and gene silencing
 Antisense RNA
 RNA interference
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 singlestranded RNA that is complementary to
a mRNA strand transcribed within a
cell. That regulates the gene
expression on the translation level.
Mechanism of antisense RNA
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。
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 posttranscriptional gene silencing
(PTGS)
Mechanism of RNAi
Normal condition
in cells
Basic process of RNAi
 Screening RNAi site to a target mRNA
 Synthesis of siRNA corresponding to a
target mRNA
 Construction of recombinant vector
carrying a siRNA
 Transfecting cell with recombinant
vector
 Analysis of target mRNA and it’s
protein
RNAi basic
process