Problem-solving test: Conditional gene targeting using the Cre/loxP recombination system

Authors


Abstract

Terms to be familiar with before you start to solve the test: gene targeting, knock-out mutation, bacteriophage, complementary base-pairing, homologous recombination, deletion, transgenic organisms, promoter, polyadenylation element, transgene, DNA replication, RNA polymerase, Shine-Dalgarno sequence, restriction endonuclease, polymerase chain reaction, primer, transcription, SV40 virus, malignant transformation, spacer region, gene expression, agarose gel electrophoresis, ethidium bromide staining, Southern-blotting, probe, reporter gene. © 2013 by The International Union of Biochemistry and Molecular Biology, 41(6):445–449, 2013

Abbreviations
Cre

causes recombination

loxP

locus of X-ing over P1

bp

base-pair

PCR

polymerase chain reaction

MCQ

multiple-choice question

Gene Targeting

Targeted gene manipulation is a powerful technique to analyze gene function. Creation of transgenic animals with selective inactivation of one of their specific genes has been widely used in the last two decades to study the role of genes. (The concept of such knock-out mutations was presented earlier in this series of problem-solving tests [1].) One caveat of this method is that knocking-out vital genes essential for early development may cause embryonic death and thus the power of the technique in detailed functional analysis of such genes becomes limited. To solve this problem, techniques for conditional manipulation of specific genes have been developed: gene targeting or altered expression of the specific gene occurs in certain cell lineages of the experimental animal, but vital embryonic functions remain unaffected. One of these methods uses the Cre/loxP recombination system [2].

Cre (for causes recombination) is a site-specific recombinase enzyme of bacteriophage P1 that recognizes short sequences called loxP (for locus of X-over P1) in the bacteriophage genome. LoxP sequences are able to interact with each other by complementary base-pairing; the Cre protein binds to the base-paired loxP elements, cleaves them in the middle and catalyzes homologous recombination between them. If two loxP elements are on the same DNA molecule and have the same orientation, Cre-catalyzed recombination leads to the deletion of sequences between the loxP sites (Fig. 1). Since endogenous loxP sequences do not exist in mammalian DNA, conditional deletion can occur in DNA regions into which loxP sites had previously been introduced.

Figure 1.

Conditional gene deletion by the Cre/loxP system. LoxP sites flanking a target sequence (A) in genomic DNA may bind to each other by complementary base-pairing (B). The Cre protein binds to this structure, cuts through the middle of both elements and causes crossing over. As a result, the target sequence is released from the genomic DNA as a circular DNA molecule (C).

The following experiment provides an example for the use of this sophisticated method [3].

The Experiment

The feasibility of the Cre/loxP recombination system was tested in a transgenic mouse model. Two sets of transgenic mice were created in this experiment. In one of them (designated hCMV-cre) the coding region of the cre gene was linked to the strong promoter of the human cytomegalovirus DNA. A polyadenylation element, (A)n, was also added to the distal end of cre (Fig. 2A.).

Figure 2.

The structure of DNA constructs used to generate transgenic mice. A: hCMV-cre. B: mαA-spacer-T (for details see the text; short vertical arrows indicate BamHI restriction endonuclease cleavage sites; short horizontal arrows correspond to primers used in PCR reactions of Figs. 3 and 4, arrowheads point to the 3′-ends of the primers).

Five-choice Completion

(This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.)

  1. ____ Why is it necessary to add the virus promoter to the cre gene?
    1. Without that the transgene cannot be replicated in mammalian cells
    2. Bacteriophage promoters do not bind eukaryotic RNA polymerases
    3. The transcript will be supplied with a Shine-Dalgarno sequence required for ribosome binding
    4. B and C
    5. A, B, and C
  2. ____ What is the consequence of adding the polyadenylation element?
    1. The transcript will be cleaved downstream of this sequence
    2. The absence of polyadenylation element could hamper efficient transcription termination
    3. The released mRNA becomes polyadenylated
    4. B and C
    5. A, B, and C

The other recombinant transgene (mαA-spacer-T) consisted of the promoter of a murine αA-crystallin gene (mαA, active in the lens cells of the eyes only), the gene of SV40 virus coding for the large tumor antigen (expression of the large T protein malignantly transforms mammalian cells), and a spacer fragment inserted between the promoter and the large T gene (Fig. 2B). The spacer, besides other sequences, contained a polyadenylation element and was flanked by two loxP sites. What happens if this transgene is introduced into a mammalian lens cell?

Relationship Analysis

(This type of question consists of a sentence with two main parts: an assertion and a reason for that assertion. Select

  1. if both assertion and reason are true statements and the reason is a correct explanation of the assertion;
  2. if both assertion and reason are true statements but the reason is not a correct explanation of the assertion;
  3. if the assertion is true but the reason is a false statement;
  4. if the assertion is false but the reason is a true statement;
  5. if both assertion and reason are false statements.)

3 .____Transfection of murine lens cells with the mαA-spacer-T transgene leads to malignant transformation of the cells, BECAUSE the large T antigen is actively expressed from the construct.

Five-choice Completion

(This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.)

  • 4. ____ What would be the size of transcript produced from this construct in lens cells?
    1. Less than 1,300 nucleotides
    2. 1,300 nucleotides
    3. 4,800 nucleotides
    4. 3,500 nucleotides
    5. There would be no transcription

hCMV-cre transgenic mice were then crossed with mαA-spacer-T transgenic animals and the offsprings were analyzed for the presence of the transgenes (Figs. 3 and 4). Polymerase chain reaction (PCR) was used to detect the cre and large T genes (Fig. 3A) and to study the loxP region (Fig. 3B) in three offsprings of a litter. (Under the PCR conditions used regions larger than 500 bp were not efficiently amplified.) The size of the PCR products were determined by electrophoresis in ethidium bromide containing agarose gels (Figs. 3A and 3B). The mαA-spacer-T region was further analyzed by Southern-blotting (Fig. 3C).

Figure 3.

Genotypes of a mαA-spacer-T x hCMV-cre litter. A: PCR analysis of tail DNA samples of three offsprings using cre (see Fig. 2A) and large T specific primers (see Fig. 2B); (B) PCR analysis of tail DNA samples using the primer pair binding to DNA sequences flanking the spacer fragment (see Fig. 2B.); (C) Southern-blot analysis of BamHI-digested tail DNA samples using the mαA-spacer-T fragment (see Fig. 2B) as a probe (bp, base-pair).

Figure 4.

PCR analysis of the loxP regions in transgenic mice (for details see the text).

Using the PCR approach, the transgene genotypes of a large number of mice involved in the experiment were determined; the results are summarized in Table 1.

Table 1. Transgenic mouse lines and occurrence of lens tumors
Genotype of transgenic miceNumber of miceNumber of mice with lens tumor
mαA-spacer-T860
hCMV-cre>1000
mαA-spacer-T/hCMV-cre1515
Wild type (no transgene)110

Finally, the loxP region of tail and eye tissue samples of two transgenic mice was analyzed using the primer pair shown in Fig. 2B. The results of this PCR analysis are shown in Fig. 4.

Study the results presented in Table 1 and Figs. 3 and 4 and solve the following multiple-choice questions (MCQs).

Five-choice Completion

(This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.)

  • 5. ____ Which of the following statements are true for offspring 2 in Fig. 3?
    1. It contains the cre gene
    2. The large T protein is expressed in its lens cells
    3. The large T protein is expressed in its fibroblasts
    4. A and B
    5. A, B, and C

Four-choice Association

(In this type of question a set of lettered headings is followed by a list of numbered words or phrases.

Select:

  1. if the word or phrase is associated with A only;
  2. if the word or phrase is associated with B only;
  3. if the word or phrase is associated with A and B;
  4. if the word or phrase is associated with neither A nor B.)
    1. Offspring 1 in Fig. 3
    2. Offspring 3 in Fig. 3
    3. Both of them
    4. Neither of them
  • 6. ____ Its genome contains a single loxP site.
  • 7. ____ Its genome contains the cre gene.
  • 8. ____ The Cre protein is expressed in its cells.
  • 9. ____ Its genome contains multiple copies of the mαA-spacer-T construct.
  • 10. ____ This animal has a high chance of developing lens tumor.
  • 11. ____ This mouse is double-transgenic (mαA-spacer-T/hCMV-cre).

Experiment Analysis

(The following statements are related to the information presented in the description of the experiment. Based on the information given, select

  1. if the statement is supported by the information given;
  2. if the statement is contradicted by the information given;
  3. if the statement is neither supported nor contradicted by the information given.)
    • 12. ____ The hCMV promoter is active in lens cells only.
    • 13. ____ The hCMV promoter is active in fibroblasts only.
    • 14. ____ The large T protein is expressed in the fibroblasts of mαA-spacer-T transgenic mice.
    • 15. ____ The large T protein is expressed in the lens cells of mαA-spacer-T transgenic mice.
    • 16. ____ Targeted deletion of the spacer region by Cre recombinase was achieved in all double-transgenic mice of Table 1.

Correct Answers

  • 1. B          9. B
  • 2. E          10. C
  • 3. E          11. C
  • 4. A          12. B
  • 5. A          13. B
  • 6. A          14. B
  • 7. C          15. B
  • 8. C          16. A

Explanations

The aim of this experiment was to demonstrate that a loxP-flanked sequence can be removed from the genome of mice in vivo by the expression of bacteriophage P1 Cre recombinase. To this end, a reporter gene coding for SV40 virus large T antigen was used. In the mαA-spacer-T transgenic mice the reporter gene is dormant, since the polyadenylation element in the spacer triggers premature transcription termination, the large T protein is thus not expressed and does not cause tumor (MCQ 2: E; MCQ 3: E; MCQ 4: A; MCQ 14: B; MCQ 15: B). Cells of transgenic hCMV-cre mice express the Cre recombinase (MCQ 1: B). Offsprings in mαA-spacer-T x hCMV-cre crosses are usually double-transgenic (MCQ 11: C): their genomes contain both transgenes. (Offspring 2 in Fig. 3 is an exception: it lacks the mαA-spacer-T transgene /MCQ 5: A/) Offspring 1 contained a single mαA-spacer-T transgene from which the spacer region was eliminated: this is indicated by the appearance of the 220 bp PCR product in Fig. 3B and by the absence of the 1,300 bp spacer fragment on the Southern-blot (MCQ 6: A). Cre-catalyzed excision of the spacer region took place in offspring 3 of Fig. 3 as well (note the 220 bp PCR product in Fig. 3B), but this animal acquired several copies of the transgene and some of them retained the spacer region (note the 1,300 bp band on the Southern-blot, Fig. 3C; MCQ 9: B). As demonstrated by the results in Fig. 3 both offspring 1 and 3 carry the hCMV-cre transgene (MCQ 7: C), express the Cre protein (MCQ 8: C) that deletes the loxP-flanked, inhibitory spacer fragment. Since the large T protein is expressed from the lens cell specific crystallin promoter, both mice will die of eye-cancer (MCQ 10: C), provided they are not sacrificed during the experiment.

Tissue specificity in this experiment is thus provided by the lens-specific mαA promoter. The hCMV promoter is active in lens and tail cells as well (see samples 3 and 4 in Fig. 4; MCQ 12: B; MCQ 13: B), but eye-tumors develop in double-transgenic mice only (MCQ 16: A).

Ancillary