Gene Cloning

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The Production and Uses of Transgenic Organisms 401 ions of toxic metals such as mercury or cadmium: essentially, they act as “molecular sponges” which sequester these metals from other proteins which could be damaged by them. The proteins have no other known function. Mice contain two genes for metallothionein proteins (MTI and MTII) which are quite close (about 6 kb apart), so the knockout construct was made by inserting oligonucleotides containing stop codons into exons for both genes and cloning a neo marker into the DNA between the two genes. After selection for resistance to neomycin and gancylcovir, as described above, PCR with oligonucleotides for both the genes was used to detect clones where both genes now contained the stop codons. Mice were generated from these cells, and, after identification of a line where the double knockout was transmitted to progeny, a homozygous line was established. This line was normal for growth and fertility, but, as predicted, was much more sensitive to the toxic metal ion Cd 2+ than the wild-type. The procedure is illustrated in Figure 12.18. Q12.9. How could you produce mice which are “double knockouts”; that is, carrying disruptions at two different genetic loci? Knockout organisms produced following cloning by nuclear transfer The success of the method described above for targeting specific loci has the potential drawback that it depends on the availability of a suitable line of ES cells plus the technology to get these cells back into a developing embryo and to get the embryo to grow to term. It is also the case that, as the initial progeny produced are mosaics (with some cells containing the novel gene disruption and some not), a second generation must be bred to get animals which carry the transgenes in all their cells. This is acceptable in the case of mice, which have a very short generation time, but for livestock animals which may take several years to reproduce it imposes a long delay on these types of experiments. Yet there is much interest in making knockout livestock, for various reasons. An alternative route to obtaining transgenic livestock with specific genes disrupted is that used in the production of Dolly the sheep, the first cloned mammal, namely cloning by nuclear transfer. Cloning by nuclear transfer does not, in itself, involve any deliberate genetic manipulation. The principle is very simple (although the practice is very difficult): nuclei from a cell line of an organism are isolated and injected into a cell that has had its own nucleus removed, and the cell is allowed to divide and then implanted into a surrogate mother. If the cell develops successfully to a mature animal, that animal should be effectively a clone of the animal from which the nucleus first originated. (However, remember the point made earlier that gene expression in animals generated from nuclear transfer may for some genes vary quite considerably from control animals.)
402 Gene Cloning Chromosome Construct A MT1 TAA neo MTII TAA tk TAA neo TAA 1. Stem cells isolated and transformed with construct with both MT genes mutated 2. Selection of G418 R gancyclovir R cells selects for homologous recombinants 3. Transformed cells confirmed by PCR then mixed with early embryos (at 32 cell stage) 4. Embryo implanted into host mothers 5. Mice develop to term 6. Offspring bred to identify those with MT knockout in germ line 7. Homozygous knockouts produced by crossing heterozygotes, and tested for Cd 2+ sensitivity by injecting cadmium salt solution 8. A normally non-lethal concentration of CdSO 4 is lethal to the knockout homozygotes Figure 12.18 Transgenic mice constructed to be deficient in metallothionein genes are more sensitive to cadmium poisoning. Clearly, if the cell line which is used as the nuclear donor is first genetically altered to disrupt a gene, then this gene disruption will be present in the cells of the mature organism that is produced. This has been the basis for several reports of knockout sheep and pigs, and although the technology is still very new, it is of such potential importance that we’ll consider it briefly here. One application of gene knockout technology in livestock is the possibility of producing pigs whose organs could be used for transplants into humans (this process of transplanting organs from one species into another is called xenotransplantation). There are many problems associated with such possibilities, including the fact that when such transplants
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Gene Cloning
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Published by: Taylor & Francis Grou
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vi Contents 3.14 Designing PCR Prim
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viii Contents 8.3 Identifying Eukar
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1 Introduction 1.1 The Beginning of
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Introduction 3 which also conveys a
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Introduction 5 thought, or the brin
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2 Genome Organization Learning outc
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Genome Organization 9 and not quite
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Genome Organization 11 both of whic
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Genome Organization 13 relative com
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Genome Organization 15 Box 2.2 Inte
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Genome Organization 17 Table 2.2 Hu
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Genome Organization 19 close to oth
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Genome Organization 21 individual g
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slower rate than junk DNA. (To be m
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Genome Organization 25 kb 0 2 4 6 8
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Genome Organization 27 cloning pred
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Genome Organization 29 (a) 1 2 3 4
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Genome Organization 31 interphase a
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Genome Organization 33 A2.3. Chromo
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3 Key Tools for Gene Cloning Learni
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Key Tools for Gene Cloning 37 repli
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Key Tools for Gene Cloning 39 Box 3
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Key Tools for Gene Cloning 41 (a) (
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Key Tools for Gene Cloning 43 then,
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Key Tools for Gene Cloning 45 Figur
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Key Tools for Gene Cloning 47 Clear
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Key Tools for Gene Cloning 49 that
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Key Tools for Gene Cloning 51 same
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3.9 More About Vectors Detecting su
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Key Tools for Gene Cloning 55 in th
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Key Tools for Gene Cloning 57 Q3.13
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Key Tools for Gene Cloning 59 alway
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Key Tools for Gene Cloning 61 Figur
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Key Tools for Gene Cloning 63 (a) C
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Key Tools for Gene Cloning 65 Box 3
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Key Tools for Gene Cloning 67 simpl
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Key Tools for Gene Cloning 69 (a) 5
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Key Tools for Gene Cloning 71 92 92
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Key Tools for Gene Cloning 73 polym
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Key Tools for Gene Cloning 75 (a) T
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Key Tools for Gene Cloning 77 DNA s
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Key Tools for Gene Cloning 79 Q3.7.
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Key Tools for Gene Cloning 81 A3.15
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Key Tools for Gene Cloning 83 A3.20
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86 Gene Cloning you extract DNA fro
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88 Gene Cloning Box 4.1 Preparation
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90 Gene Cloning which is 6.3 Mb, to
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92 Gene Cloning Also, if all the fr
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94 Gene Cloning (a) (b) Bacterial s
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96 Gene Cloning (a) Insertion vecto
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98 Gene Cloning The strictly define
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100 Gene Cloning BamHI Ap r Tet r p
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102 Gene Cloning parB Cm r parA rep
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104 Gene Cloning (a) TTTTT AAAAA TT
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106 Gene Cloning Box 4.3 Converting
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108 Gene Cloning of your gene is. A
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110 Gene Cloning A4.2. Extract chro
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112 Gene Cloning A4.8. See Section
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114 Gene Cloning Q4.15. What are th
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116 Gene Cloning dystrophin gene is
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118 Gene Cloning it is still a form
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120 Gene Cloning One obvious proble
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122 Gene Cloning The main difficult
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124 Gene Cloning (a) Labeled single
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126 Gene Cloning N terminus Phe Val
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128 Gene Cloning Box 5.3 Types of D
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130 Gene Cloning Box 5.4 Methods fo
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132 Gene Cloning the DNA probe. Bot
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134 Gene Cloning a b c d e f g h 1
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136 Gene Cloning S. cerevisiae, and
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138 Gene Cloning Q5.7. Would you ex
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140 Gene Cloning use your genomic c
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142 Gene Cloning diseases, where th
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144 Gene Cloning (a) Replicative Ta
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146 Gene Cloning where the transpos
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148 Gene Cloning DNA fragment in th
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150 Gene Cloning Bacteria transform
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152 Gene Cloning Wild-type gene Mut
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154 Gene Cloning molecule using PCR
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156 Gene Cloning digesting chromoso
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158 Gene Cloning + + Ac Avr Avr cf-
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160 Gene Cloning which are very clo
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162 Gene Cloning Box 6.3 Restrictio
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164 Gene Cloning Initial region clo
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166 Gene Cloning Box 6.4 Nonsense S
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168 Gene Cloning 6.10 Cloning of th
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170 Gene Cloning are two possible o
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172 Gene Cloning Isolation of the t
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174 Gene Cloning Two approaches wer
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176 Gene Cloning synthesize DNA de
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178 Gene Cloning Box 7.1 Denaturing
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180 Gene Cloning G A T C Figure 7.4
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182 Gene Cloning although other con
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184 Gene Cloning A large DNA fragme
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186 Gene Cloning chain reaction, fl
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188 Gene Cloning rounds of amplific
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190 Gene Cloning Contig 2 Clone 12
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192 Gene Cloning λ Clone Scaffold
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194 Gene Cloning Genomic DNA (a) Cl
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196 Gene Cloning therefore sequence
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198 Gene Cloning Tag sequence CGTGT
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200 Gene Cloning Genomic DNA (a) Ad
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202 Gene Cloning bases. The light s
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204 Gene Cloning A7.3. (a) The dide
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8 Bioinformatics Learning outcomes:
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Bioinformatics 209 Table 8.1 The ge
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Bioinformatics 211 5' accgcgcatggtg
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Bioinformatics 213 Correlation Scor
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Bioinformatics 215 AGG T R A G T C
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Bioinformatics 217 conjunction with
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Bioinformatics 219 (c) V S V K L Q
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Bioinformatics 221 Tiny P Small Ali
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Bioinformatics 223 Matrix: EBLOSUM6
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Bioinformatics 225 Box 8.2 DNA and
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Bioinformatics 227 (a) DB:ID Source
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Bioinformatics 229 (a) Sequences pr
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Bioinformatics 231 list is also of
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Bioinformatics 233 this relates to
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Bioinformatics 235 common functiona
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Bioinformatics 237 These two regula
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Bioinformatics 239 three-dimensiona
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Bioinformatics 241 Neisseria mening
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Bioinformatics 243 A8.1. The triple
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Bioinformatics 245 Q8.11. Which gro
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Bioinformatics 247 EMBL Nucleotide
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250 Gene Cloning What sorts of stud
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252 Gene Cloning 9.2 Requirements f
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254 Gene Cloning (a) The lac promot
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256 Gene Cloning lac: CCAGGCTTTACAC
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258 Gene Cloning phage (such as T7)
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260 Gene Cloning 9.4 Some Problems
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262 Gene Cloning Figure 9.6 Inclusi
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264 Gene Cloning Box 9.1 Translatio
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266 Gene Cloning Box 9.2 Signal Seq
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268 Gene Cloning Ribosome Cytosol P
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270 Gene Cloning Other yeast specie
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272 Gene Cloning (turned on by an i
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274 Gene Cloning 9.6 A Final Word A
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276 Gene Cloning study, as they wil
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10 Gene Cloning in the Functional A
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Gene Cloning in the Functional Anal
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316 Gene Cloning genomic sequences
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318 Gene Cloning the first exon of
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320 Gene Cloning Box 11.1 End Label
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322 Gene Cloning mRNA 5' 3' 3' 5' S
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324 Gene Cloning mRNA 5' 3' 3' 5' G
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326 Gene Cloning cloning step. RACE
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328 Gene Cloning Prepare RNA from c
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330 Gene Cloning Gene A condition 1
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332 Gene Cloning (a) (b) (c) Immobi
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334 Gene Cloning tat and one in whi
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336 Gene Cloning Box 11.3 Examples
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338 Gene Cloning (a) MCS for promot
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340 Gene Cloning important regulato
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342 Gene Cloning (a) F DP DPA DNA-t
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344 Gene Cloning DNA alone DNA-prot
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346 Gene Cloning [FNR] GA - + -90 -
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348 Gene Cloning (a) 1 2 3 4 5 Prom
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452 Gene Cloning Vector A self-repl
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454 Gene Cloning Capillary electrop
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456 Gene Cloning EST see expressed
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458 Gene Cloning Metallothionein, 3
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460 Gene Cloning Protein addition o
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462 Gene Cloning Transcription, ide
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Gene Cloning

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