1. Gene cloning, expression
and functional study
基因克隆，表达及功能研究

2. vectors Cloning vectors: 克隆载体 to clone a gene in a vector
Expression vectors: 表达载体
to express a gene from a vector
Integration vectors: 整合载体
to integrate a gene in a genome through a vector

3. Cloning vectors 1 Plasmid vecters 2 Bacteriophage vectors
3 Cosmids & BACs
4 Eukaryotic vectors

4. Cloning vectors: allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level.
expression vectors: allowing the exogenous DNA to be inserted, stored, and expressed.

5. A plasmid vector for cloning
Contains an origin of replication, allowing for replication independent of host’s genome.
Contains Selective markers: Selection of cells containing a plasmid
twin antibiotic resistance
blue-white screening
Contains a multiple cloning site (MCS)
Easy to be isolated from the host cell.

6. Twin antibiotic resistance screening
-Screening by insertional inactivation of a resistance gene
Ampr
Tcr
ori
pBR322
B X B B
Tcr
Ampr
Ampr X B
ori
ori
Ampicillin resistant? yes yes
Tetracycline resistant? No yes

7. Replica plating: transfer of the colonies from one plate to another using absorbent pad or Velvet (绒布).
transfer of colonies
+ampicillin
+ ampicillin
+ tetracycline
these colonies have bacteria with recombinant plasmid

8. Blue white screening
Screening by insertional inactivation of the lacZ gene
Lac promoter
MCS (Multiple cloning sites,
多克隆位点）
Ampr
pUC18
(3 kb)
lacZ’
ori
The insertion of a DNA fragment interrupts the ORF of lacZ’ gene, resulting in non-functional gene product that can not digest its substrate x-gal.

9. Recreated vector: blue transformants
Recombinant plasmid containing inserted DNA: white transformants
Recreated vector (no insert)
Recombinant plasmid (contain insert)
back

10. Multiple cloning sites
Multiple restriction sites enable the convenient insertion of target DNA into a vector
Ampr
ori
pUC18
(3 kb)
MCS (Multiple cloning sites,
多克隆位点）
Lac promoter
lacZ’
…ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA…
. T h rA s n S er S e r Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser…
EcoRI
SacI
KpnI
SmaI
XmaI
BamHI
XbaI
SalI
HincII
AccI
PstI
SphI
Lac Z

11. A plasmid vector for gene expression
Expression vectors: allowing the exogenous DNA to be inserted, stored and expressed.
Promoter and terminator for RNA transcription are required.
Intact ORF and ribosomal binding sites (RBS) are required for translation.
Include：(1) bacterial expression vectors, (2) yeast expression vectors, (3) mammalian expression vector

12. An bacterial expression vector
T7 promoter
RBS
Start codon
MCS
Ampr
Transcription terminator
T7 expression vector
ori
An bacterial expression vector

13. A yeast expression vector
MCS
A yeast expression vector

14. Bacteriophage vector Two examples: λ phage bacteriophageλ
λ replacement vector
M13 phage
M13 phage vector
Cloning in M13
Hybrid plasmid-M13 vectors

15. λ phage viruses that can infect bacteria. 48.5 kb in length
Linear or circular genome (cos ends)
Lytic phase (Replicate and release)
Lysogenic phase (integrate into host genome)

16. Cloning large DNA fragments
(Eukaryotic Genome project)
Analysis of eukaryotic genes and the genome organization of eukaryotes requires vectors with a larger capacity for cloned DNA than plasmids or phage .
Human genome (3 x 109 bp): large genome and large gene demand vectors with a large size capacity.
Genomic library VS cDNA library

17. Cosmid vectors
Utilizing the properties of the phage l cos sites in a plasmid vector.
A combination of the plasmid vector and the COS site which allows the target DNA to be inserted into the l head.
The insert can be kb

18. Formation of a cosmid clone
Digestion
Ligation
C) Packaging and infect

19. (yeast artificial chromosome)
YAC vectors
(yeast artificial chromosome)
Accommodates genomic DNA fragments of more than 1 Mb, and can be used to clone the entire human genome, but not good in mapping and analysis.

20. Essential components of YAC vectors :
Centromers (CEN), telomeres (TEL) and autonomous replicating sequence (ARS) for proliferation in the host cell.
ampr for selective amplification and markers such as TRP1 and URA3 for identifying cells containing the YAC vector in yeast cells.
Recognition sites of restriction enzymes (e.g., EcoRI and BamHI)

21. YAC Cloning

22. BAC vectors 细菌人工染色体 1. More stable than YAC 2. Capacity is 300-350 kb
3. One to two copies in each cell
4. Easy to handle
5. More popular in genomic
mapping

23. I1 Genomic libraries I1-1 Representative gene libraries
Gene libraries and screening
I1 Genomic libraries
I1-1 Representative gene libraries
I1-2 Size of library
I1-3 Genomic DNA
I1-4 Vectors

24. (made from cDNA- copy of mRNA)
I1 Genomic libraries
Gene library: a collection of different DNA sequence from an organism, each of which has been cloned into a vector for ease of purification, storage and analysis.
Genomic libraries
(made from genomic DNA)
Gene library
cDNA libraries
(made from cDNA- copy of mRNA)

25. --- Contain all the original sequences
I1 Genomic libraries
I1-1 Representative gene libraries
--- Contain all the original sequences
Missing original sequence
Certain sequences have not been cloned.
Example: repetitive sequences lacking restriction sites
Too long for the vector used
2. Library does not contain sufficient clones

26. I1-2 Size of library (ensure enough clones)
I1 Genomic libraries
I1-2 Size of library (ensure enough clones)
must contain a certain number of recombinants for there to be a high probability of it containing any particular sequence
The formula to calculate the number of recombinants:
ln (1-P)
N =
ln (1-f)
P: desired probability
f : the fraction of the genome in one insert

27. I1 Genomic libraries
For example :for a probability of 0.99 with insert sizes of 20 kb these values for the E.coli (4.6×106 bp) and human (3×109 bp) genomes are :
N E.coli= = 1.1 ×103
ln( )
ln[1-(2×104/4.6×106)]
ln(1-0.99)
Nhuman= = ×105
ln[1-(2 ×104/3 ×109)]
These values explain why it is possible to make good
genomic libraries from prokaryotes in plasmids where
the insert size is 5-10kb ,as only a few thousand
recombinants will be needed.

28. I1-3 Genomic DNA libraries
I1 Genomic libraries
I1-3 Genomic DNA libraries
eukaryotes
Purify genomic DNA
Fragment this DNA : physical shearing and restriction enzyme digestion
prokaryotes
Clone the fragments into vectors

29. Eukaryotes :prepare cell nuclei
I1 Genomic libraries
To make a representative genomic libraries ,
genomic DNA must be purified and then
broken randomly into fragments that are
correct in size for cloning into the chosen vector.
Purification of genomic DNA :
Eukaryotes :prepare cell nuclei
remove protein, lipids and other unwanted macro-
molecules by protease digestion and phase extraction.
Prokaryotes :extracted DNA directly from cells

30. pipeting, mixing or sonicaion
I1 Genomic libraries
Break DNA into fragments randomly:
Physical shearing :
pipeting, mixing or sonicaion
Restriction enzyme digestion:
partial digestion is preferred to get a greater lengths of DNA fragments.

31. Selection of restriction enzyme
I1 Genomic libraries
Selection of restriction enzyme
Ends produced (sticky or blunt) &
The cleaved ends of the vector to be cloned
Sau3A: 5’-/GATC-3’, less selectivity
BamH1: 5’-G/GATCC
Whether the enzyme is inhibited by DNA modifications (CpG methylation in mammals
Time of digestion and ratio of restriction enzyme to DNA is dependent on the desired insert size range.

32. I1-4 Vectors Vectors Plasmid phageλ cosmid YAC I1 Genomic libraries
According to genome’s size,we can select a proper vector to construct a library .
Vectors Plasmid phageλ cosmid YAC
insert (kb)
The most commonly chosen genomic cloning vectors
are λ relacement vectors which must be digested with
restriction enzymes to produce the two λ end fragment
or λ arms between which the genomic DNA will be
digested

33. λ phage vector in cloning
Long (left)
arm
short (right)
arm
cos
Exogenous DNA
(~20-23 kb)
cos
Long (left)
arm
short (right)
arm
cos
cos
Exogenous DNA
(~20-23 kb)

34. λ replacement vector cloning
0.preparation of arm and genomic inserts
2. Packing with a mixture of the phage coat proteins and phage DNA-processing enzymes
Ligation
3. Infection and formation of plaques
Library constructed

35. I 2 cDNA libraries Gene libraries and screening
I2-1 mRNA isolation, purification
I2-2 Check theRNA integrity
I2-3 Fractionate and enrich mRNA
I2-4 Synthesis of cDNA
I2-5 Treatment of cDNA ends
I2-6 Ligation to vector

36. No cDNA library was made from prokaryotic mRNA.
I 2 cDNA libraries
cDNA libraries
No cDNA library was made from prokaryotic mRNA.
Prokaryotic mRNA is very unstable
Genomic libraries of prokaryotes are easier to make and contain all the genome sequences.

37. cDNA libraries are very useful for eukaryotic gene analysis
I 2 cDNA libraries
cDNA libraries
cDNA libraries are very useful for eukaryotic gene analysis
Condensed protein encoded gene libraries, have much less junk sequences.
cDNAs have no introns  genes can be expressed in E. coli directly
Are very useful to identify new genes
Tissue or cell type specific (differential expression of genes)

38. I 2 cDNA libraries
I2-1 mRNA isolation
Most eukaryotic mRNAs are polyadenylated at their 3’ ends
oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA.
AAAAAAAAAAn
5’ cap

39. I 2 cDNA libraries

40. Three methods to isolate mRNA.
I2 cDNA libraries
Three methods to isolate mRNA.
1.Traditionally method was done by pass a preparation of total RNA down a column of oligo (dT)-cellulose
2.More rapid procedure is to add oligo(dT) linked to magnetic beads directly to a cell lysate and ‘pulling out’ the mRNA using a strong magnet
3.Alternative route of isolating mRNA is lysing cells and then preparing mRNA-ribosome complexes on sucrose gradients

41. I2-2 Check the mRNA integrity
I2 cDNA libraries
I2-2 Check the mRNA integrity
Make sure that the mRNA is not degraded. Methods:
Translating the mRNA : use cell-free translation system as wheat germ extract or rabbit reticulocyte lysate to see if the mRNAs can be translated
Analysis the mRNAs by gel elctrophoresis: use agarose or polyacrylamide gels

42. Enrichment: carried out by hybridization
I2 cDNA libraries
I2-3 Cloning the particular mRNAs
Is useful especially one is trying to clone a particular gene rather to make a complete cDNA library.
Fractionate on the gel: performed on the basis of size, mRNAs of the interested sizes are recovered from agarose gels
Enrichment: carried out by hybridization
Example: clone the hormone induced mRNAs (substrated cDNA library)

43. Second strand synthesis: best way of
I2 cDNA libraries
I2-4 Synthesis of cDNA :
First stand synthesis: materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs (Fig 1.1)
Second strand synthesis: best way of
making full-length cDNA is to ‘tail’ the 3’-end of the first strand and then use a complementary primer
to make the second. (Fig2.1)

44. I2 cDNA libraries Fig 1.1 The first strand synthesis mRNA 5’ AAAAA-3’
HO-TTTTTP-5’
Reverse transcriptase
Four dNTPs
mRNA 5’
AAAAA-3’ 3’
TTTTTP-5’
cDNA
Terminal transferase
dCTP
mRNA 5’
AAAAA-3’
3’-CCCCCCC
TTTTTP-5’
cDNA
Alkali (hydrolyaes RNA)
Purify DNA oligo(dG)
5’-pGGGG-OH
3’-CCCCCCC
TTTTTP-5’
cDNA
Klenow polymerase or reverse
Transcriotase Four dNTPs
5’-pGGGG
-3’
3’-CCCCCCC
TTTTTP-5’
Duplex cDNA
Fig The first strand synthesis

45. Single strand-specific nuclease
Duplex cDNA
5’-pGGGG
-3’
3’-CCCCCCC
TTTTTp-5’
Single strand-specific nuclease
5’-pGGGG
-3’
3’-CCC
TTTTTp-5’
Klenow polymerase
treat with E.coRI methylase
5’-pGGGG
-3’
3’-CCCC
TTTTTp-5’
Add E.colRI linkers
using T4 DNA ligase
HO-CCG/AATTCGG-3’
3’-GGCTTAA/GCC-OH
HO-CCGAATTCGGGGGG
CCGAATTCGG-3’
3’-GGCTTAAGCCCCCC
TTTTTGGCTTAAGCC-OH
E.colRI digestion
5’-pAATTCGGGGGG
CCG-3’
3’-CCCCCCC
TTTTTGGCTTAAp-5’
Ligate to vector and transfom
Fig Second strand synthesis

46. I2-5 Treatment of cDNA ends
I2 cDNA libraries
I2-5 Treatment of cDNA ends
Blunt and ligation of large fragment is not efficient, so we have to use special acid linkers to create sticky ends for cloning.
The process :
Move protruding 3’-ends(strand-special nuclease)
Fill in missing 3’ nucleotide (klenow fragment of
DNA polyI and 4 dNTPs)
Ligate the blunt-end and linkers(T4 DNA ligase)
Tailing with terminal transferase or using adaptor molecules
Restriction enzyme digestion (E.coRI )

47. I2-6 Ligation to vector The process : I2 cDNA libraries
Any vectors with an E.coRI site would suitable
for cloning the cDNA.
The process :
Dephosphorylate the vector with alkaline
phosphatase
Ligate vector and cDNA with T4 DNA ligase
(plasmid or λ phage vector)

48. I3 Screening procedures
Gene libraries and screening
I3 Screening procedures
I3-1 Screening
I3-2 Colony and plaque hybridization
I3-3 Expression screening
I3-4 Hybrid arrest and release
I3-5 Chromosome walking (repeat screening)

49. I3-1 Screening I3 Screening procedures
The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library .
Using nucleic acid probe to screen the library based on hybridization with nucleic acids.
Analyze the protein product.

50. I3 Screening procedures
Screening libraries
Searching the genes of interest in a DNA library
Hybridization to identify the interested DNA or its RNA product
Radiolabeled probes which is complementary to a region of the interested gene
Probes:
An oligonucleotide derived from the sequence of a protein product of the gene
A DNA fragment/oligo from a related gene of another species
Blotting the DNA or RNA on a membrane
Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane

51. I3-2 Colony and plaque hybridization
I3 Screening procedures
I3-2 Colony and plaque hybridization
Transfer the DNA in the plaque or colony to a
Nylon or nitrocellulose membrane
Phage DNA bind to
the membrane directly
Bacterial colonies must be lysed to
release DNA on the membrane surface.
Hybridization (in a solution
Containing Nucleic acid probe)
(Alkali treatment)
X-ray film(radio-
actively labeled )
antibody or enzyme
(modified nucleotide labeled
Wash to remove unhybri-
dization probe and visualize
Line up the hybridizated region or
repeated hybridization

52. I3 Screening procedures
Transfer to nitrocellulose
or nylon membrane
Keep master
plate
Select positive
from master plate
Denature DNA(NaOH)
Bake onto membrane
Probe with 32p-labled DNA
complementary to
gene of interest
Expose to film
Screening by plaque hybridization

53. I3-3 Expression screening
I3 Screening procedures
I3-3 Expression screening
Identify the protein product of an interested gene
Protein activity
Western blotting using a specific antibody

54. Expression screening (1)
I3 Screening procedures
Expression screening (1)
If the inserts are cloned into an expression sites, it may be expressed. Therefore, we can screen for the expressed proteins. However, this screening may miss the right clone
Example: the EcoRI site of lgt11 vector. The inserted genes have one in six change (1/6) to be in both the correct orientation (2 possibilities;  ) and reading frame (three possibilities; three nucleotide code XXX).

55. Expression screening (2)
I3 Screening procedures
Expression screening (2)
Antibodies can be used to screen the expression library.
The procedure has similarities to the plaque
hybridization protocol.
‘Plaque lift’ ( taken by placing a
membrane on the dish of plaque)
Immersed in a solution of the antibody
Detected by other antibodies
Repeat cycles of screening
to isolate pure plaques

56. 基因表达 1. Prokaryotic expression vector 原核表达载体
2. Baculovirus expression vector
昆虫杆状病毒表达载体
3. Mammalian expression vector
哺乳动物表达载体
4. Adenoviral and retroviral vector
腺病毒及逆转录病毒表达载体

57. Prokaryotic expression vector
原核表达载体
GST-fusion
6xHis-fusion
GST
HIS

58. 基因功能研究 Overexpression in cells 超表达，观察表型 RNAi 干扰技术
Yeast two hybrid system 酵母双杂交等技术寻找与目的基因相关的蛋白
Protein expression and antibody preparation表达蛋白与抗体制备
Localization of protein 蛋白在细胞中的定位

59. The end